CN1313061C - Sex sorting method of animal sperma - Google Patents
Sex sorting method of animal sperma Download PDFInfo
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- CN1313061C CN1313061C CNB021041415A CN02104141A CN1313061C CN 1313061 C CN1313061 C CN 1313061C CN B021041415 A CNB021041415 A CN B021041415A CN 02104141 A CN02104141 A CN 02104141A CN 1313061 C CN1313061 C CN 1313061C
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Abstract
The present invention relates to a sex sorting method of animal sperms. The present invention aims to provide a sex sorting method of animal sperms. The method can ensure the vitality of sorted sperms on the basis that the sorting precision is ensured after being used for sorting sperms. The method has low cost, and is suitable for commercial and large-scale production. The technical scheme of the present invention comprises the following steps: 1, carrying out fluorescence dye after the original semen of an animal is diluted by a dilution liquid; 2, sorting the semen by a cell flow type instrument under the condition of 4 DEG C to obtain the sperms of a required type. The sperms obtained by sorting can be frozen and stored for standby after being concentrated under the condition of 4 DEG C. for ensuring the vitality of the sorted sperms, both the dilution liquid and a sheath liquid for sorting contain at least one of sodium citrate, fruit sugar and yolk. The method of the present invention obviously enhances the vitality and the quality of the sorted and frozen sperms after being used for sorting animal sperms, and has the advantages of short sorting time and low sorting expense. The sorting method is simple, is suitable for large-scale industrial production, and has huge commercial value.
Description
Technical field
The present invention relates to a kind of sex sorting method of animal sperm.
Background technology
Artificial sex controll is carried out in breeding to domestic animal, is the hope that people dream of, and can improve raiser's economic benefit to greatest extent.With milk cattle cultivating already is example, and milk cattle cultivating wishes to obtain heifer per family, and with traditional mating system, will have half filial generation is bull.5% of the too late heifer value of the value of general stot, thus the reproductive capacity of half milch cow and sizable financial resources, material resources and time are wasted.The domestic animal of other kind also has similar situation.Therefore, the artificial control of domestic animal sex, to domestic animal, particularly breeding and the production of the higher domestic animal of added value have profound significance.Use domestic animal sex controll technology to avoid the breeding of undesired certain sex domestic animal, not only can reduce the cost of livestock reproduction greatly, also can accelerate the speed of animal breeding effectively, thereby obtain remarkable economic efficiency.
The artificial control technology of domestic animal sex is more, and the link that relates generally to comprises the fetus level, differentiates that as ultrasonic sex embryo's level is as embryo's sex identification and sperm level, as the external sperm sex sorting.The above two all carry out sex identification after the embryo forms, therefore, cause 50% embryo and educated the wasting of resources of parent in 50% year.Simultaneously, embryo's external sex identification not only can cause wound to embryo self, can also cause the pregnancy rate of parent to reduce.So the sex sorting of external sperm and utilize the sperm after the sorting to carry out artificial insemination is produced the filial generation of expectation sex, is livestock reproduction research worker's a important topic.
In decades, people have attempted several different methods and have separated the sperm (male) of taking X-bearing sperm (female) and carrying Y chromosome, but these methods mostly lack good reliability, repeatability and accuracy.The fluidic cell sorting technology is generally acknowledge at present reliable, high and the high efficiency sperm method for separating of accuracy, its principle is according to taking the different of X-bearing sperm and the sperm DNA content that carries Y chromosome, pass through fluorescence staining, make X sperm fluorescence volume after being excited greater than the fluorescence volume of y sperm, by the detection and the capture systems of pinpoint accuracy, collection has the sperm of desired characteristic, thereby reaches the purpose (United States Patent (USP): 5,135,759 5,985,216 6071689) of sorting.But the weak point of this technology is, as commercially producing, its expense is too high, and the vigor loss after the sperm sorting simultaneously is bigger, influence insemination effect.
Summary of the invention
The sex sorting method that the purpose of this invention is to provide a kind of animal sperm, can guarantee by the motility of sperm after the sorting, and cost is low on the basis that guarantees sharpness of separation with the sperm of this method sorting, is applicable to commercialization large-scale production.
Technical scheme of the present invention is: a kind of sex sorting method of animal sperm may further comprise the steps:
1) with the former seminal fluid of animal with diluted after, carry out fluorescence staining;
2) under 4 ℃ of conditions, seminal fluid is carried out sorting, need to obtain the sperm of type with cell streaming instrument.
The sperm that sorting obtains can be frozen standby after concentrating under 4 ℃ of conditions.
In order to ensure by the vigor of sorting sperms, described diluent is for being added with at least a material in sodium citrate, fructose and the egg yolk on the basis of basic buffer, the percentage by weight that described sodium citrate, fructose and egg yolk account for buffer is respectively 0.2-1.6%, 0.5-1.5% and 5-25%.
Wherein, preferred diluent is for being added with the sodium citrate of 0.2-1.6%, fructose and the 5-25% egg yolk (accounting for the percentage by weight of buffer) of 0.5-1.5% on the basis of basic buffer.
In order to make solution environmental and the cryopreserving liquid of sperm during sorting close, to preserve motility of sperm, it is at least a material that is added with on the basis of basic buffer in sodium citrate, fructose and the egg yolk that described branch is selected sheath fluid for use, and the percentage by weight that described sodium citrate, fructose and egg yolk account for buffer is respectively 0.2-1.6%, 0.5-1.5% and 2-10%.
Wherein, preferred sheath fluid is for being added with the sodium citrate of 0.2-1.6%, the fructose of 0.5-1.5% and the egg yolk (accounting for the percentage by weight of buffer) of 2-10% on the basis of basic buffer.
Used basic buffer is various in above-mentioned diluent and the sheath fluid, so long as the buffer that can be accepted by the animal cell all can, for example can be phosphate buffer, normal saline or desk-top buffer etc.
Utilize method of the present invention that animal sperm is carried out sorting, component by buffer and sheath fluid in control environment temperature and the assorting room, the energy expenditure of sperm and buffer have been reduced to the equilibrated influence of sperm, shortened after the sorting to the freezing required time, on the basis that keeps existing sorting degree of accuracy and separation velocity, obviously improved the vigor and the quality of frozen semen after the sorting, distinguishing period obviously shortens, sorting charge significantly reduces, method for separating is more simple, be applicable to large-scale industrial production, have huge commercial value.
Compare with existing animal sperm sorting technology, method of the present invention has the outstanding advantage of following several respects:
1, existing sorting technology is after fluorescence staining, and all operations are all at room temperature carried out.Because the assorting room required time is longer, and at room temperature, sperm is in strong active state always, power consumption is big, can cause the death of part sperm.Simultaneously sperm is before freezing, need under 4 ℃ of conditions balance 2-3 hour preserving the due vigor of sperm, so distinguishing period is longer.The present invention is dexterously by the temperature that controls environment, make the sperm after the fluorescence staining, before sorting, divide choose and sorting after all place 4 ℃ of environment, the activity and the energy expenditure of sperm have been reduced on the one hand, make sperm after fluorescence staining, promptly enter 4 ℃ of necessary poised states simultaneously, significantly shortened sperm after collection to the frozen required time, promptly improved sperm survival rate, shortened distinguishing period again.
2, in existing animal sperm sorting technology, the diluent of former seminal fluid has the Hepes buffer that contains bovine serum albumin (BSA), the lactic acid of containing, acetone acid, albuminous Tai Shi buffer are arranged, Tris buffer that contains BSA etc. is arranged, but experimental result shows, sperm is placed in above-mentioned buffer for a long time, can obviously influence its vigor.The present invention has creatively designed the diluent that contains sodium citrate, fructose and egg yolk, make motility of sperm during sorting in, can keep the vigor of conventional seminal fluid basically.
3, in existing animal sperm sorting technology, dividing selects for use sheath fluid that normal saline is arranged, phosphate buffer, Tai Shi buffer etc., its composition differs bigger with cryopreserving liquid, influences keeping of motility of sperm, these buffer all need contain the bovine serum albumin of 0.1%-1.0% simultaneously, and large usage quantity, and the bovine serum albumin price is higher, causes sorting cost costliness.The present invention has designed a kind of buffer that contains sodium citrate, fructose and egg yolk as sheath fluid, makes the solution environmental of sperm during sorting, and is close substantially with cryopreserving liquid, on the one hand motility of sperm preserved, and the sorting cost is descended greatly.
The present invention will be further described below in conjunction with drawings and Examples.
Description of drawings
The sperm streaming cytological map of Fig. 1 for obtaining with method sorting of the present invention.
The sperm PCR reacted electrophoretogram of Fig. 2 for obtaining with method sorting of the present invention.
The specific embodiment
Embodiment 1, with the X sperm in the method sorting cow seminal fluid of the present invention
1, with the fresh cattle seminal fluid diluted of the present invention of gathering, the basic buffer of diluent is a phosphate buffer, and the percentage by weight of each composition is as follows in the diluent:
Sodium citrate 0.32
Fructose 0.9
Egg yolk 20
Sodium hydrogen phosphate 0.21
Sodium chloride 0.52
Penicillin 100u/ml
Streptomycin 100ug/ml
Water adds to 100ml
pH 7.4
2, carry out fluorescence staining with fluorescent dye Hoechst33342 5ug/ml, 32 ℃ of incubations 40 minutes place 4 ℃ of cold closetes;
3, under 4 ℃ of conditions, seminal fluid is carried out the sorting of X type, sheath fluid used in the assorting room (percentage by weight is basic buffer with phosphate buffer) composed as follows with FACS VANTAGE SE type (U.S. company BD production) cell streaming instrument:
Sodium citrate 0.32
Fructose 0.9
Egg yolk 5
Sodium hydrogen phosphate 0.21
Sodium chloride 0.52
Penicillin 100u/ml
Streptomycin 100ug/ml
Water adds to 100ml
pH 7.4
4, it is frozen the sperm that obtains to be concentrated the back under 4 ℃ of conditions;
5, rewarming, record survival rate and accuracy, the result shows that the survival rate of sperm is 51%, the X sperm accounts for 91%.
In this embodiment, if expect y sperm, then on cell streaming instrument, seminal fluid is carried out the sorting of Y type.
In this embodiment, can also an adding citric acid sodium in used diluent and the sheath fluid, a kind of in fructose and the egg yolk three in the material or two kinds, experimental result shows, though the survival rate and the accuracy of sperm slightly descend when adding a kind of or two kinds of materials, but statistical analysis shows that difference is remarkable (P≤0.05) not.
Embodiment 2, usefulness flow cytometry method are identified the accuracy of the inventive method sperm sorting
The sperm that will obtain with the method sorting of embodiment 1 is removed the tail of sperm with Ultrasonic Cell Disruptor, and then through fluorescence staining, analyzes its separating purity on cell streaming instrument.The result as shown in Figure 1, continuous 5 separation results show that after the X sorting, the purity of X sperm is 92.2 ± 3.1% (89-95%), after the Y sorting, the purity of y sperm is 93.5 ± 3.8% (90-96%).
Embodiment 3, usefulness PCR method are identified the accuracy of the inventive method sperm sorting
Method with embodiment 1 obtains sorting sperms.Sperm after the sorting by multistage dilution, is chosen the test tube that contains single sperm and carried out pcr amplification.Adopt BOV97m primer specificity amplification Y chromosome fragment, adopt and utilize the primer amplification cow genome group dna fragmentation of cattle 1.715 satellite DNA sequential designs to control as the positive.Pcr amplification adopts 2-3 level amplifying method, promptly after finishing 35 circulation reactions of the first order, gets half reactant liquor and adds and carry out 35 the circulation reactions in the second level in another reaction tube and amplify, and appears as effecting reaction with positive DNA contrast fragment.Analyze that the result of sperm shows that as shown in Figure 2, the X sperm accounts for 91% after the X sorting after 100 sortings, y sperm accounts for 93% after the Y sorting, illustrates that the inventive method is reliable, accurately.
Embodiment 4, different sorting temperature are to the influence of motility of sperm
Get the fresh cattle seminal fluid of collection, be divided into three groups of room temperature, 4 ℃ of sortings and 4 ℃ of not sortings, in the buffer that embodiment 1 introduces, 32 ℃ of incubations are after 40 minutes, and 4 ℃ of sortings and 4 ℃ of not sorting groups all place 4 ℃ of cold closetes, and the room temperature group places under 25 ℃ of conditions, on the cell streaming instrument that mixes up in advance, carry out the sorting of seminal fluid then, 4 ℃ of sorting group sortings under 4 ℃ of conditions, the sorting under 25 ℃ of conditions of room temperature group, 4 ℃ of not sorting groups balance under 4 ℃ of conditions.With all spermatozoa cryopreservations, rewarming writes down survival rate then after the sorting.
The result shows: the sperm survival rate of 4 ℃ of not sortings and 4 ℃ of sortings is close substantially, is respectively 51.5 ± 8.6% and 48.9 ± 9.2%.And the sperm survival rate of room temperature group is starkly lower than the former, is 40.2 ± 10.5%, and the quality of sperm behind the survival rate of ambient temperature effect sperm during the sorting and the rewarming is described.
Embodiment 5, different semen diluent are to the influence of sperm quality
Get the fresh cattle seminal fluid of collection, respectively with the HEPES liquid that contains 1%BSA, contain the tyrode's solution of 1%BSA and contain the Tris liquid of 1%BSA and embodiment 1 in phosphoric acid diluted of the present invention, 32 ℃ of incubations 40 minutes, place 4 ℃ of cold closetes, then at 4 ℃ of environment through the sorting of cell streaming instrument, concentrate and frozen all sortings after seminal fluid, rewarming writes down survival rate again.
The result shows that HEPES liquid sperm survival rate is 42.6 ± 9.8%, tyrode's solution sperm survival rate is 45.9 ± 8.2%, Tris liquid sperm survival rate is 38.9 ± 12.2%, diluent sperm survival rate of the present invention is 47.6 ± 8.9%, illustrate that the designed diluent of the present invention is not only cheap, can keep good sperm quality simultaneously.
Embodiment 6, different sheath fluid are for the influence of sorting sperms quality
Get the fresh cattle seminal fluid of collection, be divided into the tyrode's solution group that contains 1%BSA, contain the sheath fluid group of introducing among the phosphate buffer group of 1%BSA and the embodiment 1 of the present invention.According to the method for embodiment 1 with phosphoric acid diluted of the present invention, and with above-mentioned different sheath fluid under 4 ℃ of environment, through the sorting of cell streaming instrument, concentrate, frozen, rewarming then, record sperm survival rate.
The result shows, tyrode's solution group sperm survival rate is 48.6 ± 10.2%, and phosphate buffer group sperm survival rate is 43.9 ± 7.6%, and sheath fluid group sperm survival rate of the present invention is 50.8 ± 8.9%, illustrate that the design's sheath fluid is not only cheap, can keep good sperm quality simultaneously.
Claims (10)
1, a kind of sex sorting method of animal sperm may further comprise the steps:
1) with the former seminal fluid of animal with diluted after, carry out fluorescence staining;
2) under 4 ℃ of conditions, seminal fluid is carried out sorting, need to obtain the sperm of type with cell streaming instrument.
2, the sex sorting method of animal sperm according to claim 1 is characterized in that: the sperm that sorting obtains concentrates the back under 4 ℃ of conditions frozen.
3, the sex sorting method of animal sperm according to claim 1 and 2, it is characterized in that: described diluent is for being added with at least a material in sodium citrate, fructose and the egg yolk on the basis of basic buffer, the percentage by weight that described sodium citrate, fructose and egg yolk account for buffer is respectively 0.2-1.6%, 0.5-1.5% and 5-25%.
4, the sex sorting method of animal sperm according to claim 3 is characterized in that: described basic buffer is phosphate buffer, normal saline or desk-top buffer.
5, the sex sorting method of animal sperm according to claim 3, it is characterized in that: described diluent is for to be added with sodium citrate, fructose and egg yolk on the basis of basic buffer, the percentage by weight that they account for buffer is respectively 0.2-1.6%, 0.5-1.5% and 5-25%.
6, the sex sorting method of animal sperm according to claim 5 is characterized in that: described basic buffer is phosphate buffer, normal saline or desk-top buffer.
7, the sex sorting method of animal sperm according to claim 1 and 2, it is characterized in that: it is at least a material that is added with on the basis of basic buffer in sodium citrate, fructose and the egg yolk that described branch is selected sheath fluid for use, and the percentage by weight that described sodium citrate, fructose and egg yolk account for buffer is respectively 0.2-1.6%, 0.5-1.5% and 2-10%.
8, the sex sorting method of animal sperm according to claim 7 is characterized in that: described basic buffer is phosphate buffer, normal saline or desk-top buffer.
9, the sex sorting method of animal sperm according to claim 7, it is characterized in that: described sheath fluid is for to be added with sodium citrate, fructose and egg yolk on the basis of basic buffer, the percentage by weight that they account for buffer is respectively 0.2-1.6%, 0.5-1.5% and 2-10%.
10, the sex sorting method of animal sperm according to claim 9 is characterized in that: described basic buffer is phosphate buffer, normal saline or desk-top buffer.
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| Application Number | Priority Date | Filing Date | Title |
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| CNB021041415A CN1313061C (en) | 2002-03-11 | 2002-03-11 | Sex sorting method of animal sperma |
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| CNB021041415A CN1313061C (en) | 2002-03-11 | 2002-03-11 | Sex sorting method of animal sperma |
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| CN1313061C true CN1313061C (en) | 2007-05-02 |
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Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| BR0312843A (en) * | 2002-07-22 | 2005-05-10 | Xy Inc | System to process sperm cells |
| CN101014700B (en) * | 2004-03-29 | 2012-03-28 | 英格朗公司 | Sperm suspensions for sorting into x or y chromosome-bearing enriched populations |
| WO2005095960A1 (en) * | 2004-03-29 | 2005-10-13 | Monsanto Technology Llc | Use of a composition which regulates regulates oxidation/reduction reactions intracellularly and/or extracellularly in a staining or sorting process of spermatozoa |
| US20140359795A1 (en) * | 2013-05-31 | 2014-12-04 | Recombinetics, Inc. | Genetic techniques for making animals with sortable sperm |
| CN109223245B (en) * | 2018-09-26 | 2021-02-05 | 江苏农牧科技职业学院 | Artificial insemination method of breeding goose sperms suitable for refrigeration |
| CN111500530A (en) * | 2020-04-30 | 2020-08-07 | 延安大学 | Universal animal sperm sorting method and X sperm quality control method |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN86101836A (en) * | 1986-03-22 | 1987-01-24 | 中国农业科学院畜牧研究所 | Solution for dilution of cock semen |
| US5135759A (en) * | 1989-05-10 | 1992-08-04 | The United States Of America As Represented By The Secretary Of Agriculture | Method to preselect the sex of offspring |
| US5985216A (en) * | 1997-07-24 | 1999-11-16 | The United States Of America, As Represented By The Secretary Of Agriculture | Flow cytometry nozzle for high efficiency cell sorting |
| US6071689A (en) * | 1997-12-31 | 2000-06-06 | Xy, Inc. | System for improving yield of sexed embryos in mammals |
-
2002
- 2002-03-11 CN CNB021041415A patent/CN1313061C/en not_active Expired - Fee Related
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN86101836A (en) * | 1986-03-22 | 1987-01-24 | 中国农业科学院畜牧研究所 | Solution for dilution of cock semen |
| US5135759A (en) * | 1989-05-10 | 1992-08-04 | The United States Of America As Represented By The Secretary Of Agriculture | Method to preselect the sex of offspring |
| US5985216A (en) * | 1997-07-24 | 1999-11-16 | The United States Of America, As Represented By The Secretary Of Agriculture | Flow cytometry nozzle for high efficiency cell sorting |
| US6071689A (en) * | 1997-12-31 | 2000-06-06 | Xy, Inc. | System for improving yield of sexed embryos in mammals |
| CN1284128A (en) * | 1997-12-31 | 2001-02-14 | Xy公司 | Sex-speific insemination of mammals with low number of sperm cells |
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