Animals, experimental design, and management
The experiments were carried out in Jiangnan White geese. The Jiangnan White goose is a 3-line-crossed commercial white goose in China and was certified by the National Examination and Approval Committee of Domestic Animal and Poultry Breeds in 2018. They are characterized by an intermediate size, rapid early growth, good meat quality, and a strong tolerance and adaptability to coarse feed.
Two experiments were conducted to evaluate the effects of CSM on growth performance, liver redox status, and serum biochemical parameters in goslings of different ages. Both experiments were performed at Yangzhou University Experimental Farm (Gaoyou, China). Experiment 1 was performed from April to May 2019. A total of 300 1-d-old male goslings were obtained from a commercial hatchery (Changzhou Four Seasons Poultry Industry Co. Ltd., Jintan, China). The goslings were randomly divided into 5 groups, with 6 replicates per group and 10 goslings per replicate; the test period was 28 d. In experiment 2, a total of 240 28-d-old male goslings were obtained from Suqian Lihua Animal Husbandry Co. Ltd. (Shuyang, China). The goslings were randomly divided into 5 groups, with 6 replicates per group and 8 goslings per replicate; the test period was 35 d from October to November 2020.
In both experiments, five isonitrogenous and isoenergetic diets were formulated to meet or exceed the nutrient requirements of geese according to the National Research Council (NRC) [12] and previous studies on medium-sized geese from our laboratory [13–16]. A corn-soybean meal (corn-SBM) basal diet was used as the control; 25, 50, 75 or 100% of dietary protein provided by SBM was replaced with protein provided by CSM in the other 4 diet groups (7.08, 14.15, 21.23 and 28.30% CSM (FG: 780 mg/kg) in experiment 1; 5.66, 11.33, 16.99 and 22.65% CSM (800 mg/kg) in experiment 2), which are referred to as the CSM25, CSM50, CSM75, and CSM100 groups, respectively. All energy- and protein-containing ingredients (i.e., corn, SBM, CSM, rice husk, and wheat bran) were analyzed for crude protein, crude fiber, and calcium concentrations before diet formulation. The compositions of the experimental diets are listed in Tables 1 and 2.
Table 1
Feed ingredient and nutrient composition of experimental diets for d 1 to 28 (air-dry basis)
Items
|
Control1
|
CSM25
|
CSM50
|
CSM75
|
CSM100
|
Ingredient, %
|
|
|
|
|
|
Corn
|
59.50
|
61.02
|
62.55
|
64.07
|
65.60
|
Soybean meal (46.96% CP)
|
27.60
|
20.70
|
13.80
|
6.90
|
0.00
|
Cottonseed meal (46.14% CP)
|
0.00
|
7.08
|
14.15
|
21.23
|
28.30
|
Rice husk
|
3.90
|
3.17
|
2.45
|
1.72
|
1.00
|
Wheat bran
|
5.40
|
4.36
|
3.31
|
2.27
|
1.23
|
Limestone
|
1.00
|
1.07
|
1.15
|
1.23
|
1.30
|
Calcium hydrogen phosphate
|
1.16
|
1.12
|
1.08
|
1.04
|
1.00
|
DL-Methionine
|
0.14
|
0.14
|
0.13
|
0.13
|
0.12
|
L-lysine.HCl
|
0.00
|
0.04
|
0.08
|
0.11
|
0.15
|
Salt
|
0.30
|
0.30
|
0.30
|
0.30
|
0.30
|
Premix2
|
1.00
|
1.00
|
1.00
|
1.00
|
1.00
|
Total
|
100.00
|
100.00
|
100.00
|
100.00
|
100.00
|
Nutrient level3, %
|
|
|
|
|
|
ME (MJ/kg)
|
11.39
|
11.39
|
11.39
|
11.39
|
11.39
|
CP
|
19.26
|
19.26
|
19.26
|
19.27
|
19.28
|
Crude fiber
|
5.39
|
5.39
|
5.39
|
5.39
|
5.39
|
Calcium
|
0.87
|
0.87
|
0.87
|
0.87
|
0.87
|
Available phosphorus
|
0.41
|
0.41
|
0.41
|
0.41
|
0.41
|
Lysine
|
0.94
|
0.94
|
0.94
|
0.94
|
0.94
|
Methionine
|
0.40
|
0.40
|
0.40
|
0.40
|
0.40
|
Free gossypol (mg/kg)
|
0.00
|
55.19
|
110.37
|
165.56
|
220.74
|
1Control group was fed a corn-soybean meal basal diet; CSM, cottonseed meal; CSM25, CSM50, CSM75, and CSM100 indicate that 25, 50, 75, and 100% of the protein content provided by soybean meal in the control diet was replaced with CSM respectively |
2Provided per kilogram of complete diet: Vitamin A, 9000 IU; vitamin D, 3000 IU; vitamin E, 18 IU; vitamin K, 1.5 mg; vitamin B1, 0.9 mg; vitamin B2, 8 mg; vitamin B6, 3.2 mg; vitamin B12, 0.01 mg; nicotinic acid, 45 mg; pantothenic acid, 11 mg; folic acid, 0.65 mg; biotin 0.05 mg; choline, 0.35 g; Fe (as ferrous sulphate), 60 mg; Cu (as copper sulphate), 10 mg; Mn (as manganese sulphate), 95 mg; Zn (as zinc sulphate), 90 mg; I (as potassium iodide), 0.5 mg; Se (as sodium selenite), 0.3 mg |
3Calculated values |
Table 2
Feed ingredient and nutrient composition of experimental diets for d 29 to 63 (air-dry basis)
Items
|
Control1
|
CSM25
|
CSM50
|
CSM75
|
CSM100
|
Ingredient, %
|
|
|
|
|
|
Corn
|
59.10
|
60.41
|
61.73
|
63.04
|
64.35
|
Soybean meal (43.00% CP)
|
25.05
|
18.79
|
12.53
|
6.26
|
0.00
|
Cottonseed meal (46.14% CP)
|
0.00
|
5.66
|
11.33
|
16.99
|
22.65
|
Rice husk
|
6.76
|
6.48
|
6.21
|
5.93
|
5.65
|
Wheat bran
|
5.30
|
4.80
|
4.30
|
3.80
|
3.30
|
Limestone
|
1.00
|
1.06
|
1.12
|
1.17
|
1.23
|
Calcium hydrogen phosphate
|
1.27
|
1.23
|
1.19
|
1.14
|
1.10
|
DL-Methionine
|
0.12
|
0.12
|
0.12
|
0.12
|
0.12
|
L-lysine.HCl
|
0.00
|
0.05
|
0.10
|
0.15
|
0.20
|
Choline chloride
|
0.10
|
0.10
|
0.10
|
0.10
|
0.10
|
Salt
|
0.30
|
0.30
|
0.30
|
0.30
|
0.30
|
Premix2
|
1.00
|
1.00
|
1.00
|
1.00
|
1.00
|
Total
|
100.00
|
100.00
|
100.00
|
100.00
|
100.00
|
Nutrient level3, %
|
|
|
|
|
|
ME (MJ/kg)
|
11.03
|
11.03
|
11.03
|
11.03
|
11.03
|
CP
|
17.00
|
17.00
|
17.00
|
17.00
|
17.00
|
Crude fiber
|
6.45
|
6.45
|
6.45
|
6.44
|
6.44
|
Calcium
|
0.86
|
0.86
|
0.86
|
0.86
|
0.86
|
Available phosphorus
|
0.42
|
0.42
|
0.42
|
0.42
|
0.42
|
Lysine
|
0.87
|
0.87
|
0.87
|
0.87
|
0.87
|
Methionine
|
0.37
|
0.37
|
0.37
|
0.37
|
0.37
|
Free gossypol (mg/kg)
|
0
|
45.30
|
90.60
|
135.90
|
181.20
|
1Control group was fed a corn-soybean meal basal diet; CSM, cottonseed meal; CSM25, CSM50, CSM75, and CSM100 indicate that 25, 50, 75, and 100% of the protein content provided by soybean meal in the control diet was replaced with CSM respectively |
2Provided per kilogram of complete diet: Vitamin A, 9000 IU; vitamin D, 3000 IU; vitamin E, 18 IU; vitamin K, 1.5 mg; vitamin B1, 0.6 mg; vitamin B2, 6 mg; vitamin B6, 2 mg; vitamin B12, 0.01 mg; nicotinic acid, 30 mg; pantothenic acid, 9 mg; folic acid, 0.5 mg; biotin 0.04 mg; Fe (as ferrous sulphate), 60 mg; Cu (as copper sulphate), 10 mg; Mn (as manganese sulphate), 95 mg; Zn (as zinc sulphate), 90 mg; I (as potassium iodide), 0.5 mg; Se (as sodium selenite), 0.2 mg |
3Calculated values |
The geese were raised in wire-floor pens (1.5 m × 0.9 m in experiment 1; 1.8 m × 0.9 m in experiment 2) and had ad libitum access to feed and water. Each pen was equipped with a separate feeder and 2 automatic nipple drinkers. In experiment 1, the geese were exposed to a 24-h photoperiod from d 1 to 14 and an 18-h photoperiod from d 15 to 28. The house temperature was maintained at 28 to 30℃ for the first week and decreased by 2℃ each week until the house temperature was approximately 22℃ on d 28. In experiment 2, the birds were reared under natural daylight, and the room temperature was maintained at approximately 20℃. The experimental protocol and use of animals were approved by the animal care and use committee of Yangzhou University (Yangzhou, China).
Sample collection and preparations
Birds were individually weighed on their arrival. At the end of each trial (d 28 in experiment 1; d 63 in experiment 2), goslings were weighted after a 6-h feed withdrawal, and feed consumption of each replicate pen was recorded. Average daily feed intake (ADFI), average daily gain (ADG), and the feed to gain ratio (F:G) were calculated. Mortality was recorded daily.
At the end of each trial (d 28 in experiment 1; d 63 in experiment 2), 30 goslings (1 gosling per pen) with average body weight (BW) were selected. Approximately 2 mL of blood was collected via the wing vein and centrifuged at 2,000 × g for 10 min at 4℃. Serum was collected and stored at − 20℃ until biochemical parameter analysis. Once the blood was collected, birds were exsanguinated by severing the jugular vein and carotid artery on one side of the neck. Samples of the liver (left lobes, 6 goslings/treatment) were excised, flash-frozen in liquid nitrogen, and stored at − 70℃ until further analysis.
Antioxidant capacity
The liver samples were homogenized according to a previously described method [17]. The protein concentrations of the liver homogenates were determined using a Total Protein Quantitative Assay Kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).
The scavenging abilities of the superoxide radical (O2•¯) and hydroxyl radical (OH•) and the activities of superoxide dismutase (SOD) and catalase (CAT) were measured using a 725N ultraviolet-visible spectrophotometer (INESA Scientific Instrument Co., Ltd., Shanghai, China) with respective commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The results were calculated based on the protein concentrations of liver homogenates and are expressed as units per milligram of protein for O2•¯ scavenging ability (U/mg protein) and units per gram of protein for OH• scavenging ability (U/g protein). SOD and CAT activities are expressed as units per milligram of protein (U/mg protein).
Changes in glutathione (GSH) concentration and activities of GSH-related enzymes in the liver were measured spectrophotometrically with corresponding commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The GSH concentration is expressed as milligrams per gram of protein (mg/g protein). The activities of glutathione reductase (GR), glutathione peroxidase (GSH-Px), and glutathione S-transferase (GST) were calculated based on the protein concentration and are expressed as units per milligram of protein (U/mg protein).
Reactive oxygen metabolites, malondialdehyde, and protein carbonyl concentrations
The reactive oxygen metabolite (ROM) concentrations in the liver were measured spectrophotometrically according to the d-ROM test based on Costantini and Dell’Omo [18]. The results are expressed as millimoles of hydrogen peroxide (H2O2) per gram of protein in the liver (mmol H2O2/g protein). The concentration of malondialdehyde (MDA), an index of lipid peroxidation, was evaluated by the thiobarbituric acid reactive substance reaction. The MDA assay kit was purchased from Nanjing Jiancheng Bioengineering Research Institute (Nanjing, China), and the operation method followed the manufacturer’s instructions. The MDA concentration was calculated as nanomoles per milligram of protein (nmol/mg protein). Protein carbonyl (PC) concentrations, an indicator of protein oxidation, were determined by the 2,4-dinitrophenylhydrazine reaction according to the method of Gaona-Gaona et al. [19] and are expressed as nanomoles per milligram of protein (nmol/mg protein) in the liver.
Serum biochemical parameters
Serum concentrations of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total protein (TP), albumin (ALB), globulin (GLOB), and uric acid (UA) were determined using a UniCel DxC 800 Synchron fully automatic biochemical analysis system (Beckman Coulter, Los Angeles, CA, USA).
Statistical analysis
One-way ANOVA followed by linear contrast analysis were used to analyze all data in SPSS 17.0 (SPSS Inc., Chicago, IL, USA). Each replicate pen served as the experimental unit for growth, while individual goslings served as the experimental unit for the other experimental data. Mortality data were arcsin transformed before analysis. Data are expressed as means and standard errors of the means (SEMs). Significant differences among the treatment means were determined at P < 0.05 by Duncan’s multiple range tests.