CN107821336B - Method for evaluating quality of individual and population of earthworm fry - Google Patents
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- 241001614060 Amynthas aspergillus Species 0.000 claims abstract description 50
- 210000004392 genitalia Anatomy 0.000 claims abstract description 28
- 230000000694 effects Effects 0.000 claims abstract description 15
- 238000013441 quality evaluation Methods 0.000 claims abstract description 8
- 241000237636 Pheretima Species 0.000 claims abstract description 4
- 210000001015 abdomen Anatomy 0.000 claims abstract description 4
- 241000243684 Lumbricus Species 0.000 claims description 29
- 241001233061 earthworms Species 0.000 claims description 28
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 13
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Abstract
The invention discloses a method for evaluating individual and population quality of earthworm larvae. The invention discloses a quality evaluation method of earthworm fry individuals, which comprises the following steps: observing the integrity, body color and genital girdle of the Pheretima aspergillum, and measuring the weight, body length, diameter and activity of the Pheretima aspergillum; if the individual Pheretima aspergillum is complete, the back is dark brown to purple gray, and the abdomen is light yellow brown, the genital girdle is not obvious, and the weight of the individual Pheretima aspergillum is more than or equal to 4.0g, the body length is more than or equal to 9.0cm, the diameter of the genital girdle is more than or equal to 4.5mm, and the vitality is more than or equal to 35.0s, the individual Pheretima aspergillum is judged to be a qualified individual Pheretima. The evaluation method can be used for collecting and putting earthworm larvae, and promotes the development of earthworm breeding. The evaluation method is scientific and is simple and convenient to operate.
Description
The technical field is as follows:
the invention belongs to the technical field of earthworm breeding and traditional Chinese medicine resource development, and particularly relates to a method for evaluating individual and population quality of earthworm larvae.
Background art:
earthworm, also called earthworm, is a common terrestrial animal, is cold in nature and salty in taste, and has the main effects of clearing heat, arresting convulsion, dredging collaterals, relieving asthma and promoting urination. Research shows that the earthworm has the functions of reducing blood pressure, relieving asthma, tranquilizing, clearing away heat, resisting blood coagulation, resisting thrombus, resisting cancer, strengthening immunity, etc. At present, the earthworm medicinal materials in the market of China are mainly 'Guangdong earthworm' and 'Hu earthworm', wherein the Guangdong earthworm is a Guangdong medicinal material and is generally recognized as the best quality in the medicinal material industry.
With the continuous increase and change of the medical health requirements of human beings, the demand of traditional Chinese medicine resources is more and more. As most of Chinese medicinal materials come from wild, artificial breeding of the lumbricus becomes a main source along with reduction of wild resources. Therefore, screening of earthworm seedlings meeting the specification is the first link for artificial breeding of earthworms, is beneficial to growth and propagation of earthworms and provides quality guarantee for production of earthworm medicinal materials.
The invention content is as follows:
the invention aims to overcome the defects in the prior art and provides a method for evaluating the quality of individual and population of lumbricus.
The invention aims to provide a quality evaluation method for earthworm fry individuals, which is characterized by comprising the following steps: observing the integrity, body color and genital girdle of the Pheretima aspergillum, and measuring the weight, body length, diameter and activity of the Pheretima aspergillum; if the individual Pheretima aspergillum is complete, the back is dark brown to purple gray, and the abdomen is light yellow brown, the genital girdle is not obvious, and the weight of the individual Pheretima aspergillum is more than or equal to 4.0g, the body length is more than or equal to 9.0cm, the diameter of the genital girdle is more than or equal to 4.5mm, and the vitality is more than or equal to 35.0s, the individual Pheretima aspergillum is judged to be a qualified individual Pheretima.
The body length of the Guang earthworm individual refers to the length from the genital girdle to the tail.
Preferably, the specific step of measuring the body length of the individual Pheretima aspergillum is to place the Pheretima aspergillum in 50% alcohol by volume, anaesthetize the Pheretima aspergillum until the body is in a relaxed state, and measure the length from the genital girdle to the tail.
Preferably, the measuring of the diameter of the genital girdle of the individual lumbricus is specifically to measure the diameter of the genital girdle in a resting state of the lumbricus.
Preferably, the specific measurement of the individual vitality of the lumbricus is to place the lumbricus in alcohol with the volume fraction of 50% and record the time required for anaesthetizing the lumbricus until the body is in a relaxed state.
The individual complete of the lumbricus is preferably that the lumbricus has complete organs and the individual is intact.
Before the quality evaluation of the earthworm seedlings, DNA molecular identification is carried out on the earthworms to be detected.
The DNA molecular identification of the Pheretima aspergillum comprises the following steps: extracting genome DNA of Pheretima aspergillum, designing primers according to mitochondrial CO I and 16SrRNA gene sequences of Pheretima aspergillum in the gene library, performing PCR amplification, performing electrophoresis detection on PCR products, analyzing amplification result of bands, sequencing gene sequences, performing similarity search by BLAST (basic Local Alignment search) after splicing sequencing results, searching for matched sequences, analyzing similarity between the detected sample and mitochondrial CO I and 16SrRNA gene sequences of Pheretima aspergillum in the gene library, and determining whether the sample is Pheretima aspergillum variety.
The second purpose of the invention is to provide a quality evaluation method for lumbricus fry groups, which is characterized by comprising the following steps: randomly collecting a plurality of individual earthworms, measuring the weight, body length and reproductive ring diameter and activity of the individual earthworms, calculating the average value X1 and standard deviation SV1 of the weight of the individual earthworms, the average value X2 and standard deviation SV2 of the body length of the individual earthworms, the average value X3 and standard deviation SV3 of the reproductive ring diameter of the individual earthworms, the average value X4 and standard deviation SV4 of the activity of the individual earthworms, and respectively calculating the dispersion coefficients CV1, CV2, CV3 and CV4 of the weight, body length, reproductive ring diameter and activity of the individual earthworms according to the average value and standard deviation of the weight, body length, reproductive ring diameter and activity of the individual earthworms; if the conditions that X1 is more than or equal to 5.5g, CV1 is less than or equal to 0.28, X2 is more than or equal to 11.0cm, CV2 is less than or equal to 0.2, X3 is more than or equal to 5.0mm, CV3 is less than or equal to 0.16, X4 is more than or equal to 42s and CV4 is less than or equal to 0.15 are met, the earthworm fry population is judged to be a qualified Guangdong earthworm fry population.
The body length of the Guang earthworm individual refers to the length from the genital girdle to the tail.
Preferably, the specific step of measuring the body length of the individual Pheretima aspergillum is to place the Pheretima aspergillum in 50% alcohol by volume, anaesthetize the Pheretima aspergillum until the body is in a relaxed state, and measure the length from the genital girdle to the tail.
Preferably, the measuring of the diameter of the genital girdle of the individual lumbricus is specifically to measure the diameter of the genital girdle in a resting state of the lumbricus.
Preferably, the specific measurement of the individual vitality of the lumbricus is to place the lumbricus in alcohol with the volume fraction of 50% and record the time required for anaesthetizing the lumbricus until the body is in a relaxed state.
The invention has the beneficial effects that: the evaluation method is scientific, simple and convenient to operate and low in requirements on experimental instruments. The evaluation method can be used for collecting and putting earthworm seedlings of the guangdong earthworms and artificially culturing the guangdong earthworms, and promotes the growth and the propagation of the guangdong earthworms.
Description of the drawings:
FIG. 1 shows the CO I sequence of Pheretima aspergillum (Pheretima aspergillum).
The specific implementation mode is as follows:
the following examples are further illustrative of the present invention and are not intended to be limiting thereof.
Example 1: DNA molecular identification of Pheretima aspergillum
Randomly collecting samples from a culture base of the Pheretima aspergillum in Chongxi province, extracting genome DNA of the Pheretima aspergillum, designing primers according to mitochondrial COI gene sequences of the Pheretima aspergillum in the gene base, performing PCR amplification, performing gene sequence sequencing on PCR products, splicing obtained gene sequence results (shown as SEQ ID NO.1 in figure 1), searching for matched sequences through BLAST (basic Local Alignment search), analyzing similarity between the detected samples and the mitochondrial COI gene sequences of the Pheretima aspergillum in the gene base, and determining the collected samples to be Pheretima aspergillum by combining morphological analysis.
Example 2:
randomly collecting 50 samples from a Luchuan county earthworm (earthworm ginseng, earthworm) breeding base, wherein the number of the samples is 1-50. All collected lumbricus have intact organs and the individual is intact, the back is dark brown to purple gray, the abdomen is light yellow brown, and the genital girdle is not obvious. The earthworm species are both subjected to morphological identification and DNA molecular identification.
1. Weight measurement of Pheretima aspegillum
The weight of 50 samples was weighed using an electronic balance, repeated 3 times, averaged, and the statistics are shown in tables 1 and 2 below:
table 1: determination of the weight of Pheretima aspergillum (unit: g)
| Numbering | Weight (D) | Numbering | Weight (D) | Numbering | Weight (D) | Numbering | Weight (D) | Numbering | Weight (D) |
| 1 | 4.2 | 11 | 8.2 | 21 | 5.8 | 31 | 6.6 | 41 | 3.5 |
| 2 | 3.1 | 12 | 6.7 | 22 | 7.5 | 32 | 8.0 | 42 | 7.1 |
| 3 | 2.2 | 13 | 6.5 | 23 | 4.3 | 33 | 5.0 | 43 | 6.4 |
| 4 | 4.6 | 14 | 3.5 | 24 | 9.9 | 34 | 6.0 | 44 | 4.7 |
| 5 | 4.4 | 15 | 4.6 | 25 | 6.3 | 35 | 5.4 | 45 | 5.8 |
| 6 | 7.7 | 16 | 6.0 | 26 | 4.3 | 36 | 6.3 | 46 | 3.6 |
| 7 | 6.8 | 17 | 6.3 | 27 | 6.3 | 37 | 5.3 | 47 | 5.4 |
| 8 | 6.7 | 18 | 5.1 | 28 | 6.8 | 38 | 5.2 | 48 | 4.8 |
| 9 | 3.4 | 19 | 6.9 | 29 | 2.6 | 39 | 7.3 | 49 | 5.8 |
| 10 | 5.8 | 20 | 5.9 | 30 | 7.4 | 40 | 5.9 | 50 | 6.4 |
Table 2: the quantity of earthworm in the weight range
Note: "[" represents greater than or equal to, ")" represents less than
2. Determination of the diameter of the gonadal girdle of Pheretima aspergillum
Using a vernier caliper to measure the diameter of the genital ring zone of the lumbricus in a quiet state, repeating the measurement for 3 times, and taking an average number, wherein the statistical results are shown in the following tables 3 and 4:
table 3: determination of the diameter of the Carpesium Adenophorae Miq (Living-quiet) (unit: mm)
| Numbering | Diameter of | Numbering | Diameter of | Numbering | Diameter of | Numbering | Diameter of | Numbering | Diameter of |
| 1 | 4.8 | 11 | 6.2 | 21 | 5.5 | 31 | 5.7 | 41 | 4.3 |
| 2 | 4.2 | 12 | 6.6 | 22 | 5.8 | 32 | 6.3 | 42 | 6.7 |
| 3 | 5.2 | 13 | 5.9 | 23 | 4.4 | 33 | 5.2 | 43 | 6.3 |
| 4 | 5.8 | 14 | 4.9 | 24 | 5.9 | 34 | 5.5 | 44 | 5.8 |
| 5 | 5.7 | 15 | 6.2 | 25 | 4.1 | 35 | 5.3 | 45 | 5.2 |
| 6 | 7.4 | 16 | 5.6 | 26 | 5.6 | 36 | 4.7 | 46 | 5.1 |
| 7 | 5.3 | 17 | 5.7 | 27 | 5.5 | 37 | 5.1 | 47 | 5.3 |
| 8 | 7.2 | 18 | 5.1 | 28 | 6.1 | 38 | 4.4 | 48 | 5.0 |
| 9 | 3.1 | 19 | 5.2 | 29 | 4.7 | 39 | 6.1 | 49 | 4.7 |
| 10 | 5.3 | 20 | 4.6 | 30 | 6.8 | 40 | 5.1 | 50 | 5.4 |
Table 4: frequency of diameter range of lumbricus and lumbricus
Note: "[" represents greater than or equal to, ")" represents less than
3. Body length measurement of Guangdong earthworm
The earthworms are placed in alcohol with the volume fraction of 50 percent, after the earthworms are anesthetized until the body is in a relaxed state, the length from the genital girdle to the tail part is measured by a ruler, the steps are repeated for 3 times, the average number is taken, and the statistical results are shown in the following tables 5 and 6:
table 5: body length measurement (clitellum genitalis to tail) of Pheretima aspergillum treated with 50% ethanol (unit: cm)
| Numbering | Length of | Numbering | Length of | Numbering | Length of | Numbering | Length of | Numbering | Length of |
| 1 | 13.2 | 11 | 12.7 | 21 | 9.5 | 31 | 12.2 | 41 | 8.5 |
| 2 | 10.2 | 12 | 11.2 | 22 | 13.2 | 32 | 13.5 | 42 | 11.5 |
| 3 | 7.5 | 13 | 12.5 | 23 | 10.8 | 33 | 10.1 | 43 | 11.8 |
| 4 | 8.5 | 14 | 9.2 | 24 | 16.9 | 34 | 11.3 | 44 | 10.6 |
| 5 | 10.2 | 15 | 10.7 | 25 | 11.2 | 35 | 13.4 | 45 | 13.0 |
| 6 | 12.3 | 16 | 12.0 | 26 | 9.4 | 36 | 9.8 | 46 | 6.8 |
| 7 | 15.3 | 17 | 11.6 | 27 | 12.9 | 37 | 11.5 | 47 | 11.1 |
| 8 | 13.5 | 18 | 9.5 | 28 | 11.2 | 38 | 9.6 | 48 | 12.7 |
| 9 | 7.3 | 19 | 11.1 | 29 | 6.8 | 39 | 15.5 | 49 | 11.3 |
| 10 | 13.4 | 20 | 14.1 | 30 | 12.6 | 40 | 11.4 | 50 | 12.4 |
Table 6: frequency of body length range of lumbricus
Note: "[" represents greater than or equal to, ")" represents less than
4. Evaluation of Activity of Pheretima aspergillum
The earthworms were placed in 50% alcohol by volume until the earthworms were anesthetized until the body was in a relaxed state, and the time taken for each sample to react was recorded by using a stopwatch, and the statistics are shown in the following tables 7 and 8:
table 7: activity measurement result of earthworm and earthworm (50% alcohol) (unit: s)
Table 8: vitality frequency of earthworm and earthworm
| Vitality (unit: s) | Frequency of occurrence |
| Less than 34.0 | 3 |
| [34.0-37.0) | 4 |
| [37.0-40.0) | 9 |
| [40.0-43.0) | 12 |
| [43.0-46.00) | 9 |
| [46.0-49.0) | 6 |
| [49.0-52.0) | 4 |
| Greater than 52.0 | 3 |
Note: "[" represents greater than or equal to, ")" represents less than
5. And (3) evaluating the quality of the individual and the population of the lumbricus:
1) the quality evaluation result of the individual Pheretima aspergillum comprises:
the quality of 50 samples was evaluated based on the results of measurement of the weight, diameter, length and vitality of the lumbricus shown in tables 1, 3, 5 and 7 in combination with the frequency/quantity shown in tables 2, 4, 6 and 8. All 36 Guangdong lumbricus individuals with the numbers of 1, 6-8, 10-13, 15-22, 24, 26-28, 30-34, 36, 37, 39, 40, 42-45, 47, 49 and 50 simultaneously meet the conditions that the weight of the Guangdong lumbricus individual is more than or equal to 4.0g, the body length is more than or equal to 9.0cm, the diameter of a genital girdle is more than or equal to 4.5mm and the vitality is more than or equal to 35.0s, and the 36 Guangdong lumbricus individuals are judged to be qualified Guangdong lumbricus individuals (see table 9).
Table 9: quality evaluation result of earthworm and earthworm fry individual
Calculating the average value X1 of the weight of 50 earthworms, the standard deviation SV1 of 5.7g, the standard deviation SV1 of 1.53 and the dispersion coefficient CV1 of 0.268 according to the measurement results of the weight, the diameter of the genital ring area, the length of the body and the vitality of the earthworms in the tables 1, 3, 5 and 7; the average value X2 of the body length is 11.37cm, the standard deviation SV2 is 2.15, and the dispersion coefficient CV2 is 0.189; the average value X3 of the diameter of the genital belt ring is 5.4mm, the standard deviation SV3 is 0.82, and the coefficient of variation CV3 is 0.15; the average value of the activity X4 is 42.9s, the standard deviation SV4 is 6.21, and the dispersion coefficient CV4 is 0.148. Meanwhile, the conditions that X1 is more than or equal to 5.5g, CV1 is less than or equal to 0.28, X2 is more than or equal to 11.0cm, CV2 is less than or equal to 0.2, X3 is more than or equal to 5.0mm, CV3 is less than or equal to 0.16, X4 is more than or equal to 42s and CV4 is less than or equal to 0.15 are met, so that the earthworm fry population of the Guangdong earthworm in Guangdong earthworm breeding bases in Luchuan county is judged to be qualified Guangdong earthworm.
Claims (8)
1. The quality evaluation method of the earthworm fry individual is characterized by comprising the following steps: observing the integrity, body color and genital girdle of the Pheretima aspergillum, and measuring the weight, body length, diameter and activity of the Pheretima aspergillum; if the individual Pheretima aspergillum is complete, the back is dark brown to purple gray, and the abdomen is light yellow brown, the genital girdle is not obvious, and the weight of the individual Pheretima aspergillum is more than or equal to 4.0g, the body length is more than or equal to 9.0cm, the diameter of the genital girdle is more than or equal to 4.5mm, and the vitality is more than or equal to 35.0s, the individual Pheretima aspergillum is judged to be a qualified individual Pheretima; the specific measurement of the individual vitality of the Pheretima aspergillum is to place the Pheretima aspergillum in alcohol with a volume fraction of 50%, and record the time required for anesthesia until the body is in a relaxed state.
2. The method for evaluating the quality of the lumbricus individuals according to claim 1, wherein the length of the lumbricus individuals refers to the length from the genital girdle to the tail.
3. The method for evaluating the quality of the individual Pheretima aspergillum according to claim 1, wherein the measuring the length of the individual Pheretima aspergillum is performed by placing Pheretima aspergillum in 50% alcohol, and measuring the length from the genital tract to the tail after anesthetizing until the body is in a relaxed state.
4. The method for evaluating the quality of the individual Pheretima aspergillum according to claim 1, wherein the measuring the diameter of the genital ring of the individual Pheretima aspergillum is performed in a resting state of Pheretima aspergillum.
5. The quality evaluation method for lumbricus fry groups is characterized by comprising the following steps: randomly collecting a plurality of individual earthworms, measuring the weight, body length and reproductive ring diameter and activity of the individual earthworms, calculating the average value X1 and standard deviation SV1 of the weight of the individual earthworms, the average value X2 and standard deviation SV2 of the body length of the individual earthworms, the average value X3 and standard deviation SV3 of the reproductive ring diameter of the individual earthworms, the average value X4 and standard deviation SV4 of the activity of the individual earthworms, and respectively calculating the dispersion coefficients CV1, CV2, CV3 and CV4 of the weight, body length, reproductive ring diameter and activity of the individual earthworms according to the average value and standard deviation of the weight, body length, reproductive ring diameter and activity of the individual earthworms; if the conditions that X1 is more than or equal to 5.5g, CV1 is less than or equal to 0.28, X2 is more than or equal to 11.0cm, CV2 is less than or equal to 0.2, X3 is more than or equal to 5.0mm, CV3 is less than or equal to 0.16, X4 is more than or equal to 42s and CV4 is less than or equal to 0.15 are met simultaneously, judging that the earthworm fry population is a qualified Guangdong earthworm fry population; the specific measurement of the individual vitality of the Pheretima aspergillum is to place the Pheretima aspergillum in alcohol with a volume fraction of 50%, and record the time required for anesthesia until the body is in a relaxed state.
6. The method of claim 5, wherein the length of the Pheretima aspergillum individuals is from the genital girdle to the tail.
7. The method for evaluating the quality of the earthworm fry group of claim 5, wherein the measuring the length of the individual body of the earthworm is carried out by placing the earthworm in 50% alcohol by volume, and measuring the length from the genital tract to the tail of the earthworm after the earthworm is anesthetized until the body is in a relaxed state.
8. The method for evaluating the quality of the earthworm fry group of claim 5, wherein the measuring the diameter of the genital girdle of the earthworm individual is specifically to measure the diameter of the genital girdle under the resting state of the earthworm.
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| PCT/CN2018/121592 WO2019091497A1 (en) | 2017-11-07 | 2018-12-17 | Earthworm juvenile individual and group quality evaluation methods |
| AU2018364223A AU2018364223A1 (en) | 2017-11-07 | 2018-12-17 | Quality evaluation methods for individual and population of matured guang-dilong earthworms |
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| JP2001242156A (en) * | 2000-02-25 | 2001-09-07 | Toshiba Corp | Method and its monitoring support system for water quality monitoring |
| CN1785380A (en) * | 2005-12-09 | 2006-06-14 | 贵州益佰制药股份有限公司 | Quality control method of Chinese medicinal preparation |
| CN1844912A (en) * | 2006-01-24 | 2006-10-11 | 李振国 | Earthworm fingerprint spectrum establishment method and medicinal earthworm identification method |
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| JPS5381393A (en) * | 1976-12-18 | 1978-07-18 | Zenichi Kawashima | Method for producing earthworms for feeding and medical use and earthworm excremental soil for fertilizer by utilizing droppings of swine* chicken0and cattle etc* and wastes from processes for process |
| CN107656027B (en) * | 2017-09-04 | 2019-12-06 | 广东省生物资源应用研究所 | Method for evaluating quality of earthworm seedlings |
| CN107821336B (en) * | 2017-11-07 | 2021-05-11 | 广东省生物资源应用研究所 | Method for evaluating quality of individual and population of earthworm fry |
| CN108684613B (en) * | 2018-04-11 | 2020-11-06 | 广东省科学院动物研究所 | Pollution-free breeding method for lumbricus |
| CN108782467B (en) * | 2018-06-12 | 2020-11-03 | 广东省科学院动物研究所 | Method for breeding earthworm seedlings |
-
2017
- 2017-11-07 CN CN201711100812.3A patent/CN107821336B/en active Active
-
2018
- 2018-12-17 AU AU2018364223A patent/AU2018364223A1/en not_active Abandoned
- 2018-12-17 WO PCT/CN2018/121592 patent/WO2019091497A1/en active Application Filing
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| JP2001242156A (en) * | 2000-02-25 | 2001-09-07 | Toshiba Corp | Method and its monitoring support system for water quality monitoring |
| CN1785380A (en) * | 2005-12-09 | 2006-06-14 | 贵州益佰制药股份有限公司 | Quality control method of Chinese medicinal preparation |
| CN1844912A (en) * | 2006-01-24 | 2006-10-11 | 李振国 | Earthworm fingerprint spectrum establishment method and medicinal earthworm identification method |
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| 动物类中药材质量评价研究概况及分析;程樱 等;《中国药事》;20170430;第31卷(第4期);第381-388页 * |
| 地龙种质资源与品质评价研究;吴文如;《中国博士学位论文全文数据库》;20080430;第88页第2段 * |
| 地龙规范化生产的研究概况;李薇 等;《中草药》;20050930;第36卷(第9期);第1419-1422页 * |
Also Published As
| Publication number | Publication date |
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| AU2018364223A1 (en) | 2019-06-13 |
| CN107821336A (en) | 2018-03-23 |
| WO2019091497A1 (en) | 2019-05-16 |
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