Some standard content:
Cs 65. 150
National Standard of the People's Republic of China
CB19163-2003bZxz.net
2003-06-04 Issued
Bullfrog
2003-12-01 Implementation
People's Republic of China
General Administration of Quality Supervision, Inspection and Quarantine
Chapter 5 and Chapter 7 of this standard are mandatory, and the rest are recommended. Appendix B of this standard is a normative appendix and Appendix A is an informative appendix. This standard is proposed by the Ministry of Agriculture of the People's Republic of China. This standard is sponsored by the Yangtze River Fisheries Research Institute of the Chinese Academy of Fisheries Sciences. The drafting units of this standard are: Hunan Agricultural University, Hunan Provincial Animal Husbandry and Fisheries Bureau. The main drafters of this standard are: Gan Xiaoqing, Gan Yu, Xiao Keyu, Wang Xuguang, Zhang Yuanzhi, Chen Liban, Song Qing. GD19163—2003
GB19163—2003
This standard gives the main morphological and structural characteristics, growth and reproduction, genetic characteristics, and detection methods of bullfrog (Ranacatesheiana Shaw).
This standard applies to the detection and identification of bullfrog germplasm. 2 Normative references
The following documents are cited in this standard as clauses of this standard. For dated references, all subsequent amendments (excluding errata) or revisions are not applicable to this standard. However, the parties who reach an agreement based on this standard are encouraged to study whether the latest versions of these documents can be used. For undated references, the latest versions are applicable to this standard. GB17716 Qingyu
3 Name and classification
3.1 Scientific name
Bullfrog Runa catesbeiana Shaw.
3. 2 Classification position
Anura, Ranidae, Rana. 4 Terms and definitions
The following terms and definitions apply to this standard. 4.1
Head length
The length from the tip of the snout to the rear edge of the maxillomandibular joint. 4.2
Bead width
The distance between the left and right jaw joints.
Eye diameter
Orbital diameter.
Drum diameter
Drum diameter.
Forearm length
The length from the joint to the end of the third finger.
Finger (toe) order
The order of the fingers (toes) from short to long.
GB 19163—2003
Nuptial pad pad
The more developed tumor on the inner side of the first toe of male frogs, which is related to reproductive activities. 4.8
hindleg lengthhindlegJength
The length from the cloaca to the end of the fourth toe.
fout lengih
The length from the inner process to the end of the fourth toe. 4. 10
hoxly length
The length from the end of the tail to the end of the tail.
5Main morphological and structural characteristics
5.1External morphological characteristics
5. 1. 1 Appearance
The body is short and thick, the head is wide and flat, slightly triangular, and there is a pair of round eardrums located at the back of the head. The color of the eardrums is basically the same as the color of the back of the head. There are obvious skin folds from the back of the eyes to the top of the eardrums. The skin is relatively smooth, and the back is slightly rough with very fine skin. There are 4 fingers on the forelimbs, no claws, and the finger patterns are A, II, and A. The female frog has a well-developed nude inside the first finger, which increases significantly during the breeding season. The frog has no nude pads: the hind limbs are particularly developed - 5 toes, full webbed + webbed incomplete to the toe tip, the toe sequence is I, II, V, II, I. The body color varies with the living environment, age, sex, and feeding conditions. Usually the head is green, the back is green-brown, yellow-brown or black, with irregular spots or stripes of varying depths, and the abdomen is grayish white with dark gray stripes. The lower face of mature male frogs is golden yellow, and the abdomen of female frogs is the same color. The appearance of bullfrogs is shown in Figure 1: 1. Dorsal view 5.1.2 Measurable traits Figure 1 Appearance of bullfrog (unit) h. Ventral view 1-2 years old, body length 10.3 cm-16, 1 c11. Weight 175, 2-548. 7 The proportion of measurable traits of bullfrogs with actual measurements is shown in Table 1. 2 Body length/head length 3.093 10.247 2. 981±0.243
5.2 Internal structural characteristics
Head width/head length
1.162±0.096
1.168±0.089
Table 1 Ratios of measured measurable traits
Drum diameter/eye diameter
0,912±0.08
1. 338 1 0. 154
Hint thigh length/body length
1. 4671 0.078
1.475±0.102
There is a pair of internal vocal sacs in the pharynx of the pheasant frog, opening into the oropharyngeal cavity; the female frog has no vocal sacs. 5.2.2 Spinal column
Foot length/forearm length
1.609 0.119
1,613±0.115
1 cervical vertebra, 7 sacral vertebrae, 1 sacral vertebrae, 1 coccygeal bone. It is a staggered body with concave in front and convex in back. 5.2.3 Reproductive system
GB19163—2003
Toe width
1.770±0.242
1.708 1 0.244
Male has a pair of light yellow oval testes, with degenerate Mullerian ducts. Female has a pair of cystic ovaries, which contain yellow early eggs and extremely black in animals and extremely milky white in plants. There is a finger-shaped fat body in front of the gonads. It is white or light yellow before sexual maturity and yellow after sexual maturity. Its size varies with seasons and nutritional status.
Growth and reproduction
6.1 Growth
The measured values of body length and weight of bullfrogs in different age groups are shown in Table 2. The growth equation and the relationship between body length and weight are shown in Appendix A. Table 2 The measured values of body length and weight of bullfrogs in different age groups Months/age
Companion length/em
Body length/name
6.2 Reproduction
6.2.1 Sexual maturity age and weight
115,73
± 17. 56
1-2 years old. Female frogs are more than 350 g, and male frogs are more than 300 g. 6.2.2 Spawning season
The northern region is from May to August, Hunan, Chaobei and other provinces are from April to September, and Dong and Fujian provinces are from March to November. The suitable water temperature for spawning is 20℃-30℃
6.2.3 Spawning frequency and type
The sexually mature frog can lay eggs once or twice a year, and mainly in spring. The eggs are ovoid, with a diameter of 1.2mm-1.5mm. The egg is very dark black and the egg is very milky white. The eggs are covered with a gelatinous membrane and can be adhered to form a single layer or a mass of eggs. 6.2.4 Egg carrying capacity
The ovarian coefficient of sexually mature female frogs in the breeding season is 12.6%~-27.6%, the absolute egg carrying capacity is (5.010.77)×10°, and the relative egg carrying capacity is (106.7±8.69) eggs/g.
7 Genetic characteristics
7.1 Cytogenetic characteristics
The number of chromosomes in bullfrog somatic cells: 2m=26. Number of arms (NF): 52. Karyotype formula, 222m+1sm. See Figure 2 for the karyotype of bullfrog.
GB 19163—2003
x×× ××
Figure 2 The karyotype of bullfrog
7.2 Biochemical genetic characteristics
10 yen
The electrophoresis enzyme spectrum of lactate dehydrogenase (LDH) isozyme of bullfrog myocardium is shown in Figure 3. The isozyme band scanning diagram is shown in Figure 4, and the activity intensity of the enzyme band is shown in Table 3.
Electrophoresis enzyme spectrum of bullfrog myocardium LH isozyme
Figure 4 Scanning of bullfrog myocardium LDH isozyme bands Figure 3 Activity intensity of bullfrog myocardium LDH isozyme bands
Relative mobility
Activity intensity/)
8 Detection method
Determination of biological traits
Measurable traits were measured with a divider and ruler readings, accurate to 0.1 cm; body weight was measured with an electronic scale, accurate to 0.1 g. Internal structural characteristics were observed by dissection and compared with standard scales. 8.2 Determination of fertility
During the breeding season,Dissect the female frog that has not laid eggs, remove the ovaries, weigh them with an electronic scale, and then weigh 1 and 0 eggs, and count the average number of eggs per gram. The number of eggs contained in the ovaries is the absolute egg-bearing capacity, and the number of eggs contained per unit body weight (g) is the relative egg-bearing capacity.
8.3 Chromosome detection
8.3.1 Specimen preparation
8 h~12 h before surgery, inject 0.1% phytohemagglutinin (PHA) 0.5 mL~1.0 mL (100 g body weight) into the lumbar cavity, and inject colchicine solution (10 g-15 g per gram body weight) into the abdominal cavity 4 h~6 h before surgery; dissect the bullfrog. Take the bone marrow from the tibia and shinbone, and put it into 0.4% potassium chloride (KCE) solution, and bathe at 37℃ for 15min~20min. Centrifuge at 1000r/min for 10min. Discard the supernatant, add 1mL of fixative (methanol and glacial acetic acid in 3:1 ratio), fix for 20min at 4°C, repeat fixation twice. Add a small amount of fixative to prepare a cell suspension; take a drop of the cell suspension, dry it on an alcohol lamp for ten minutes, immerse it in Gielsa solution (prepared according to GB 17716) and stain it for 30min, rinse off the excess stain with steam water, and air dry it to prepare a slide. 8.3.2 Chromosome counting
According to GB17716.
8.3.3 Group type analysis
Perform according to the provisions of GH17716.
8.4 Biochemical genetic analysis
8.4.1 Preparation and preservation of samples
GB 19163-2003
Dissect bovine saccharide, take the myocardium, wash off the blood with physiological fluorescent water, weigh and chop, add physiological saline at low temperature to 15 (mass concentration), centrifuge at 12000r/min for 10min at 4℃. Take the supernatant and add an equal amount of 40% sucrose solution, after spiking, mark and store in a refrigerator for 25℃.
8.4.2 Electrophoresis separation
Use a steady current and voltage electrophoresis instrument and 6% polyacrylamide gel column electrophoresis for separation. Electrophoresis is carried out at about 10℃, the sample volume is 50mL, and bromoform blue is used as an indicator. Stabilize the voltage at 280V, run the electrophoresis for 80-90 minutes, and stop when the blue reaches 1 cm from the front of the gel. 8.4.3 Staining and Fixing
After the electrophoresis is finished, take the gel and put it into the prepared staining solution, and incubate it at 37°C for 10-30 minutes until the bands are clear. Pour out the staining solution, and terminate the reaction with the fixing solution (methanol: acetic acid water is 45:12:43). The preparation of the isozyme staining solution is shown in Appendix B. 8.4.4 Scanning Determination
Scan the electrophoresis pattern with a laser scanner and conduct quantitative analysis. GB19163—2003
4.1 Growth equation for body length and weight
(Informative Appendix)
Growth equation and relationship between body length and weight
The growth equation for body length and weight is shown in formula (A.1) (A,2L 17.32[1—c-0 293 7+1.02>M, = 681, 77L1 - e: D.314 6(417.20 21 73.955 7 In the formula:
body length of bullfrog at age f, in centimeters (cm) -weight of bullfrog at age t, in grams (g); e—base of natural logarithm;
age of bullfrog in months, in years.
A,2 Relationship between body length and weight
The relationship between body length and weight of bullfrog is shown in formula (A.3), M - 0. 0:38 7 × 13.48
Wherein:
Weight of bullfrog, in grams ():
Body length of bullfrog, in centimeters (crm).
++++++++++++++++++( A, I )
+--+ A. 2 )
......( A,3)
B.1 Preparation of phosphate buffer solution (0.5mol/L) Appendix B
(Normative Appendix)
Preparation of isocyanate staining solution
GB19163—2003
Take 6.8g of potassium dihydrogen phosphate (KH.PO,) and dissolve it in about 80ml of distilled water. Adjust the pH to 7. 4 with sodium hydroxide (Na)H) solution (2mol/L). Then make up to 100% with distilled water. mL, B.2 Preparation of Nitro Blue Tetrazolium Chloride (NBT) Solution (mg/mL) Take 50 mg Nitro Blue Tetrazolium Chloride (NBT) and dilute it to 50 mL distilled water, store at 4°C. B.3 Preparation of Coenzyme I (NAD) Stock Solution (mz/ml.) Take 100 mg Coenzyme I (NAD) and dilute it to 10 mL distilled water. B.4 Preparation of Phenazine Methyl Sulfate (PMS) Stock Solution (mg/mL.) Take 50 mg Phenazine Methyl Sulfate (PMS) and dilute it to 50 mL distilled water, store at 4°C in the dark. B.5 Preparation of Sodium Chloride (NaCl) Solution (0.1 mol/L) Take 581 mg of Sodium Fluoride and dilute it to 100 mL distilled water. 3.6 Preparation of Magnesium Chloride (MgCl) Solution (0.005 mol/L) Take 100 mg of Magnesium Chloride (MgCl:6H2O) and dilute it to 100 mL distilled water. B.7 Preparation of sodium lactate solution (1 mol/L)
Take 9.35 mL of DL-sodium lactate (concentration 600 mg/mlL) and add distilled water to 50 ml. B.8 Preparation of staining solution
Take 7.5 mL of phosphodiesterone buffer, add 7.5 ml of nitro blue tetrazolium solution, 3.0 ml of coenzyme T solution, 75 mL of methyl methacrylate sulfate solution, 3.0 ml of sodium chloride solution, 3.0 ml of magnesium chloride solution, and 3.0 mL of sodium lactate solution, mix and it is ready.77L1 - e: D.314 6(417.20 21 73.955 7 In the formula:
body length of bullfrog at age f, in centimeters (cm); body weight of bullfrog at age t, in grams (g); e - base of natural logarithm;
age of bullfrog in months, in years.
A,2 Body length and weight relationship formula
The body length and weight relationship of bullfrog is shown in formula (A.3), M - 0. 0:38 7 × 13.48
In the formula:
weight of bullfrog, in grams ();
body length of bullfrog, in centimeters (crm).
++++++++++++++++++( A, I )
+--+ A. 2 )
......( A,3)
B.1 Preparation of phosphate buffer solution (0,5mol/L) Appendix B
(Normative Appendix)
Preparation of isocyanate staining solution
GB19163-2003
Dissolve 6.8g of potassium dihydrogen phosphate (KH.PO.) in about 80ml of distilled water, and adjust the pH to 7.4 with sodium hydroxide (NaOH) solution (2mol/L). Then make up to 100mL with distilled water. B.2 Preparation of nitro tetrazolium chloride (NBT) solution (mg/mL) Take 50mg of nitro tetrazolium chloride (NBT) and make up to 50tmL of distilled water, and store at 4℃. B.3 Preparation of coenzyme I (NAD) stock solution (mz/ml.) Take 100mg of coenzyme I (NAD) and make up to 10mL of distilled water. B.4 Preparation of PMS stock solution (mg/mL) Take 50 mg of PMS and dilute it in 50 ml of distilled water and store it in the dark at 4°C. B.5 Preparation of sodium chloride (NaCl) solution (0.1 mol/L) Take 581 mg of sodium fluoride and dilute it in 100 ml of distilled water. 3.6 Preparation of magnesium chloride (MgCl) solution (0.005 mol/L) Take 100 mg of magnesium chloride (MgCl: 6H,0) and dilute it in 100 ml of distilled water. B.7 Preparation of sodium lactate solution (1 mol/L)
Take 9.35 mL of DL-sodium lactate (concentration 600 mg/mlL) and add distilled water to 50 ml. B.8 Preparation of staining solution
Take 7.5 mL of phosphodiesterone buffer, add 7.5 ml of nitro blue tetrazolium solution, 3.0 ml of coenzyme T solution, 75 mL of methyl methacrylate sulfate solution, 3.0 ml of sodium chloride solution, 3.0 ml of magnesium chloride solution, and 3.0 mL of sodium lactate solution, mix and it is ready.77L1 - e: D.314 6(417.20 21 73.955 7 In the formula:
body length of bullfrog at age f, in centimeters (cm); body weight of bullfrog at age t, in grams (g); e - base of natural logarithm;
age of bullfrog in months, in years.
A,2 Body length and weight relationship formula
The body length and weight relationship of bullfrog is shown in formula (A.3), M - 0. 0:38 7 × 13.48
In the formula:
weight of bullfrog, in grams ();
body length of bullfrog, in centimeters (crm).
++++++++++++++++++( A, I )
+--+ A. 2 )
......( A,3)
B.1 Preparation of phosphate buffer solution (0,5mol/L) Appendix B
(Normative Appendix)
Preparation of isocyanate staining solution
GB19163-2003
Dissolve 6.8g of potassium dihydrogen phosphate (KH.PO.) in about 80ml of distilled water, and adjust the pH to 7.4 with sodium hydroxide (NaOH) solution (2mol/L). Then make up to 100mL with distilled water. B.2 Preparation of nitro tetrazolium chloride (NBT) solution (mg/mL) Take 50mg of nitro tetrazolium chloride (NBT) and make up to 50tmL of distilled water, and store at 4℃. B.3 Preparation of coenzyme I (NAD) stock solution (mz/ml.) Take 100mg of coenzyme I (NAD) and make up to 10mL of distilled water. B.4 Preparation of PMS stock solution (mg/mL) Take 50 mg of PMS and dilute it in 50 ml of distilled water and store it in the dark at 4°C. B.5 Preparation of sodium chloride (NaCl) solution (0.1 mol/L) Take 581 mg of sodium fluoride and dilute it in 100 ml of distilled water. 3.6 Preparation of magnesium chloride (MgCl) solution (0.005 mol/L) Take 100 mg of magnesium chloride (MgCl: 6H,0) and dilute it in 100 ml of distilled water. B.7 Preparation of sodium lactate solution (1 mol/L)
Take 9.35 mL of DL-sodium lactate (concentration 600 mg/mlL) and add distilled water to 50 ml. B.8 Preparation of staining solution
Take 7.5 mL of phosphodiesterone buffer, add 7.5 ml of nitro blue tetrazolium solution, 3.0 ml of coenzyme T solution, 75 mL of methyl methacrylate sulfate solution, 3.0 ml of sodium chloride solution, 3.0 ml of magnesium chloride solution, and 3.0 mL of sodium lactate solution, mix and it is ready.
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