AU2021103410A4 - Method for chemically inducing allotriploid abalone - Google Patents
Method for chemically inducing allotriploid abalone Download PDFInfo
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- AU2021103410A4 AU2021103410A4 AU2021103410A AU2021103410A AU2021103410A4 AU 2021103410 A4 AU2021103410 A4 AU 2021103410A4 AU 2021103410 A AU2021103410 A AU 2021103410A AU 2021103410 A AU2021103410 A AU 2021103410A AU 2021103410 A4 AU2021103410 A4 AU 2021103410A4
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- 238000000034 method Methods 0.000 title claims abstract description 20
- 230000001939 inductive effect Effects 0.000 title claims abstract description 10
- 235000013601 eggs Nutrition 0.000 claims abstract description 46
- 230000006698 induction Effects 0.000 claims abstract description 37
- 230000004720 fertilization Effects 0.000 claims abstract description 17
- 229940079593 drug Drugs 0.000 claims abstract description 14
- 239000003814 drug Substances 0.000 claims abstract description 14
- 230000035800 maturation Effects 0.000 claims abstract description 9
- 210000004508 polar body Anatomy 0.000 claims abstract description 6
- BVIAOQMSVZHOJM-UHFFFAOYSA-N N(6),N(6)-dimethyladenine Chemical compound CN(C)C1=NC=NC2=C1N=CN2 BVIAOQMSVZHOJM-UHFFFAOYSA-N 0.000 claims description 24
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 22
- 239000013535 sea water Substances 0.000 claims description 20
- 241000143510 Haliotis discus hannai Species 0.000 claims description 18
- 241000237878 Haliotis fulgens Species 0.000 claims description 15
- 230000002710 gonadal effect Effects 0.000 claims description 6
- 241000237891 Haliotidae Species 0.000 claims description 5
- 239000012535 impurity Substances 0.000 claims description 5
- 210000000287 oocyte Anatomy 0.000 claims description 5
- 230000019100 sperm motility Effects 0.000 claims description 5
- 230000003750 conditioning effect Effects 0.000 claims description 4
- 230000000638 stimulation Effects 0.000 claims description 4
- 239000011550 stock solution Substances 0.000 claims description 4
- 241001489139 Haliotis discus Species 0.000 claims description 3
- 230000037396 body weight Effects 0.000 claims description 2
- 238000007865 diluting Methods 0.000 claims description 2
- 239000006185 dispersion Substances 0.000 claims description 2
- 238000001914 filtration Methods 0.000 claims description 2
- 239000012530 fluid Substances 0.000 claims description 2
- 239000012528 membrane Substances 0.000 claims description 2
- 238000005406 washing Methods 0.000 claims description 2
- 238000009395 breeding Methods 0.000 abstract description 11
- 230000012447 hatching Effects 0.000 abstract description 10
- 208000026487 Triploidy Diseases 0.000 abstract description 9
- 230000001488 breeding effect Effects 0.000 abstract description 7
- 230000001360 synchronised effect Effects 0.000 abstract description 7
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- 235000015170 shellfish Nutrition 0.000 abstract description 5
- 208000035199 Tetraploidy Diseases 0.000 abstract description 2
- 208000036878 aneuploidy Diseases 0.000 abstract description 2
- 231100001075 aneuploidy Toxicity 0.000 abstract description 2
- 238000007599 discharging Methods 0.000 abstract description 2
- 230000008144 egg development Effects 0.000 abstract description 2
- 241000894007 species Species 0.000 description 7
- 230000018109 developmental process Effects 0.000 description 6
- 230000001418 larval effect Effects 0.000 description 5
- 241000548230 Crassostrea angulata Species 0.000 description 3
- 241001489138 Haliotis diversicolor Species 0.000 description 3
- 238000009360 aquaculture Methods 0.000 description 3
- 244000144974 aquaculture Species 0.000 description 3
- 238000009402 cross-breeding Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000000684 flow cytometry Methods 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 241001207609 Crassostrea hongkongensis Species 0.000 description 2
- 241000237874 Haliotis rufescens Species 0.000 description 2
- 208000020584 Polyploidy Diseases 0.000 description 2
- 210000000349 chromosome Anatomy 0.000 description 2
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- 231100000517 death Toxicity 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 235000014102 seafood Nutrition 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 241001441955 Argopecten irradians Species 0.000 description 1
- 241000237505 Chlamys Species 0.000 description 1
- 241000358845 Crassostrea ariakensis Species 0.000 description 1
- 241000237516 Mizuhopecten yessoensis Species 0.000 description 1
- GBOGMAARMMDZGR-UHFFFAOYSA-N UNPD149280 Natural products N1C(=O)C23OC(=O)C=CC(O)CCCC(C)CC=CC3C(O)C(=C)C(C)C2C1CC1=CC=CC=C1 GBOGMAARMMDZGR-UHFFFAOYSA-N 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- GBOGMAARMMDZGR-JREHFAHYSA-N cytochalasin B Natural products C[C@H]1CCC[C@@H](O)C=CC(=O)O[C@@]23[C@H](C=CC1)[C@H](O)C(=C)[C@@H](C)[C@@H]2[C@H](Cc4ccccc4)NC3=O GBOGMAARMMDZGR-JREHFAHYSA-N 0.000 description 1
- GBOGMAARMMDZGR-TYHYBEHESA-N cytochalasin B Chemical compound C([C@H]1[C@@H]2[C@@H](C([C@@H](O)[C@@H]3/C=C/C[C@H](C)CCC[C@@H](O)/C=C/C(=O)O[C@@]23C(=O)N1)=C)C)C1=CC=CC=C1 GBOGMAARMMDZGR-TYHYBEHESA-N 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000013020 embryo development Effects 0.000 description 1
- 230000006353 environmental stress Effects 0.000 description 1
- 238000009313 farming Methods 0.000 description 1
- 210000002149 gonad Anatomy 0.000 description 1
- 230000026109 gonad development Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 208000021267 infertility disease Diseases 0.000 description 1
- 238000009403 interspecific hybridization Methods 0.000 description 1
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K61/00—Culture of aquatic animals
- A01K61/50—Culture of aquatic animals of shellfish
- A01K61/51—Culture of aquatic animals of shellfish of gastropods, e.g. abalones or turban snails
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
- Y02A40/81—Aquaculture, e.g. of fish
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- Life Sciences & Earth Sciences (AREA)
- Environmental Sciences (AREA)
- Marine Sciences & Fisheries (AREA)
- Zoology (AREA)
- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Farming Of Fish And Shellfish (AREA)
Abstract
OF THE DISCLOSURE
The present disclosure discloses a method for chemically inducing an allotriploid abalone
and relates to a method for breeding shellfish. The method includes the steps of: step 1, selection
and accelerated maturation of broodstock; step 2, artificial induction; step 3, selection and
treatment of gametes; step 4, synchronous fertilization; step 5, drug induction; step 6, drug
removal; and step 7, hatching. Synchronous fertilization is ensured: gametes are strictly
prevented from being exposed in advance in the discharging process, absence of unintended
fertilization is confirmed through microscopic examination, and the consistency of synchronous
fertilization with fertilized egg development is guaranteed. Induction conditions are optimized:
70%-80% of the PB1 proportion of fertilized eggs in a control group is determined as a treatment
time node by constant microscopic observation, effective release of first polar bodies is
guaranteed, and tetraploids and aneuploidies are avoided. The induction rate is stable and
efficient: the triploid induction rate is 100% or close to 100%.
Description
[01] The present disclosure relates to a method for breeding shellfish, and in particular to a method for chemically inducing an allotriploid abalone.
[02] Abalone is a valuable choice rare sea food, and is hailed as the head of "the eight choice rare sea foods". As the world's largest producer of abalone, China has a total abalone yield of 148,500 tons in 2017, with an annual output value of over RMB 20 billion yuan. At present, the main abalone species cultured in China is Haliotis discus hannai, and alalones are cultured from the Yellow Sea and Bohai Sea area in northern China to the coast of Fujian Province. The aquaculture production in Fujian Province was as high as 124,400 tons (in 2017), accounting for 83.1% of the national total abalone production. However, affected by the living habits of H. discus hannai,broodstocks in the subtropical Fujian sea area have experienced large-scale deaths in summer, and the local fishermen suffered heavy losses. In order to avoid risk, farmers can only shorten the culture cycle, resulting in a variety of undesirable industrial conditions such as miniaturization of commercial specifications, weak resistance and insufficient quality. In addition, H. discus hannai in full breeding seasons will become weak after gonadal discharge, and may be prone to death under environmental stress. The above problems plaguing the development of the abalone farming industry in Fujian are urgently needed to be resolved.
[03] Cross breeding technology can achieve the purpose of germplasm improvement. H. fulgens, mainly distributed along the Pacific Ocean in the USA and Mexico, is a warm-water species and one of the main breeding abalone species in North America. Because of large size and strong high temperature adaptability thereof, since the 1990s, it has been introduced from the USA to China by WANG Zichen (1986) and XU Guojun (1989) for domestication, self production, and other studies. Since 2011, Abalone Breeding Research Group in Xiamen University has produced a new hybrid species "Lvpan Abalone" by using interspecific hybridization between H. discus hannai (y) and H. fulgens (o), which were certificated by Ministry of Agriculture and Rural Affairs of China (Accession No.: GS-02-003-2018). Compared with the parent species, Lvpan abalone (H. discus hannaiy x H. fulgens) has a significant heterobeltiosis in growth and survival, and resistance thereof is significantly enhanced. Also, it has been found that the development and maturation of the gonads of Lvpan abalone was relatively retarded, and the mortality increased in some individuals with excellent gonad development.
[04] Allopolyploid refers to a polyploid formed by chromosome doubling of a hybrid progeny produced by crossbreeding of different species. From an evolutionary perspective, allopolyploids are superior to autopolyploids; with a complete chromosome set of two or more different species, allopolyploids have a stronger natural selection advantage. Like autotriploids, allotriploids may also be infertile or poorly fertile. At present, the induction of allotriploids in shellfish is dominated by physicochemical methods, such as Crassostreaariakensis x C. gigas, Chlamys farrer x Patinopectenyessoensi, C.farrer x Argopecten irradians,C. hongkongensis x C. gigas, H. rufescens x H.fulgens, H. discus hannai x H. diversicolor(Zhang Y, Zhang Y, Wang Z, et al. Phenotypic trait analysis of diploid andtriploid hybrids from female Crassostreahongkongensis x male C. gigas[J].Aquaculture, 2014, 434:307-314.). Herein, H. rufescens (y) x H.fulgens (0) was induced by 0.5 mg/L CB, and the induction rate was 69%-91% (Hernandez-Ibarra NK, Morelos RM, Cruz P, et al. Allotriploid Genotypic Assignment in Abalone Larvae by Detection of Microsatellite-Recombinant Genotypes[J]. Journal of Heredity, 2010, 101(4):476-490.); H. discus hannai (y) x H. diversicolor(o) was induced by 0.6 mg/L CB, and the highest triploid rate was 48.11% (Hu JC, Guo BJ, Gu YJ, et al. Study on artificial culturing allotriploid abalone induction[J]. Journal of Aquaculture, 2011, 32(1):38-42.); after H. diversicolor (y) x H. discus hannai(o) was induced by 0.4 mg/L CB, the highest allotriploid percentage was 50.96% (Zhang HY, Yan ZY, Gu YJ. Allotriploid abalone induction by cytochalasin B[J]. J Jimei Univ (Nature Edition), 2010, 15(6):414-419.). However, the induction of allotriploids of H. discus hannai(y) and H. fulgens (o) has not been studied inside and outside of China. At the same time, shellfish allotriploid breeding have a series of problems, including immature induction conditions, low induction rate, unstable induction effect, and influence of induction factors on embryonic development and larval survival. Solving the above factors will be beneficial to the promotion and application of this technology.
[05] An objective of the present disclosure is to provide a method for chemically inducing an allotriploid abalone that combines cross breeding with polyploid breeding technology.
[06] The present disclosure includes the following steps:
[07] step 1, broodstock collection and conditioning;
[08] where in step 1, broodstock collection and conditioning may be specifically implemented by: selecting H. discus hannai (y) and H. fulgens (o) with excellent individual vigor and no external damage as broodstocks, and placing the abalones in parent abalone culture ponds for accelerated maturation, respectively; the broodstock may be H. discus hannai aged 2-3 years, and have a shell length of greater than 70 mm and a body weight of greater than 50 g; sex characteristics in the parent abalone culture ponds must be single, and seawater temperature in the abalone culture ponds may be 24-26°C.
[09] step 2, artificial induction;
[10] where in step 2, the artificial induction may be specifically implemented by: using a method of exposing to air for desiccation stimulation combined with ultraviolet (UV) irradiation of seawater to induce individuals with gonadal maturation until broodstock spawn and release sperms, respectively; the exposing to air for desiccation stimulation may be implemented by: after exposing to air for 60-120 min, strictly distinguishing between male and female individuals into different containers with clear labels, injecting UV-treated seawater with an irradiation intensity of 500-1,200 mWh/L UV, changing the seawater every 60 min until gamete release, and taking care to prevent unintended fertilization.
[11] step 3, selection and treatment of gametes;
[12] where in step 3, the selection and treatment of gametes may be specifically implemented by: filtering and removing impurities from the gametes in step 2 using a 150 m screen, and determining oocyte quality and sperm motility through microscopic examination; the microscopic examination may include that in an egg fluid with high quality of the microscopic examination, an egg has an intact egg membrane and a regular shape without unintended fertilization, and a sperm with excellent vigor has excellent mobility and dispersion.
[13] step 4, synchronous fertilization;
[14] where in step 4, the synchronous fertilization may be implemented by: mixing the sperms and eggs with excellent vigor and high quality determined in step 3, and dividing into a treatment group and a control group, with a mass ratio of sperm to egg of (15-20):1; the fertilization may have a temperature of 20-22°C and a salinity of 30-35.
[15] step 5, drug induction;
[16] where in step 5, the drug induction may be specifically implemented by: observing the development of fertilized eggs in the control group, and quickly adding the fertilized eggs in the treatment group into a 1.50-1.75 mg/L CB induction buffer to treat for 15-20 min or a 30-40 mg/L 6-dimethylaminopurine (6-DMAP) induction buffer to treat for 10-15 min when 50%-80% of first polar bodies appear; where the first polar bodies should be sampled and observed microscopically at intervals during the observation; the CB induction buffer may be obtained by diluting a 1 mg/mL stock solution prepared by dissolving CB in dimethyl sulfoxide (DMSO), and the 6-DMAP induction buffer may be obtained from a 50 mg/mL stock solution prepared by dissolving 6-DMAP in DMSO.
[17] step 6, drug removal; and
[18] where in step 6, the drug removal may be implemented by: placing drug-treated fertilized eggs in 1% DMSO and washing twice, 15 min each time.
[19] step 7, hatching.
[20] where in step 7, the hatching may be implemented according to a conventional hatching and post-breeding method, and veliger larval triploid rate may be determined by using a flow cytometer.
[21] The present disclosure has the following outstanding technical effects:
[22] 1. Synchronous fertilization is ensured: gametes are strictly prevented from being exposed in advance in the discharging process, absence of unintended fertilization is confirmed through microscopic examination, and the consistency of synchronous fertilization with fertilized egg development is guaranteed.
[23] 2. Induction conditions are optimized: 70%-80% of the PB1 proportion of fertilized eggs in a control group is determined as a treatment time node by constant microscopic observation, effective release of first polar bodies is guaranteed, and tetraploids and aneuploidies are avoided.
[24] 3. The induction rate is stable and efficient: the induction rate of the existing shellfish allotriploids varies between 40% and 90%, but the induction rate of the triploid of the present disclosure is 100% or close to 100%. Test results of different drugs inducing the triploid of H. discus hannai(Y) x H.fulgens (o) are shown in Table 1.
[25] Table 1
[26] Treatment Treatment Triploid rate (%) Drug concentration time (min) Test 1 Test 2 Test 3 (mg/L) CB 1.50-1.75 15-20 84.12+6.71 84.52+7.15 100.00+0.00 6-DMAP 30-40 10-15 100.00+0.00 100.00+0.00 100.00+0.00
[27] The present disclosure will be described in detail below with reference to the examples.
[28] Example 1
[29] 1) H. discus hannai and H.fulgens with excellent individual vigor and without external damage were selected as broodstocks; the H. discus hannai (Y) had an average shell length of 76.099.57 mm, a shell width of 50.664.19 mm, and a wet weight of 55.50+13.88 g; the H. fulgens (o) had an average shell length of 75.21+3.06 mm, a shell width of 51.71±1.44 mm, and a wet weight of 54.294.40 g. The broodstocks were bred in different parent abalone culture ponds, respectively, and the sex characteristics of parent abalones in each parent abalone culture pond must be single.
[30] 2) After the gonadal maturation, the broodstocks were dried in the shade for 60 min. Subsequently, male and female individuals were placed in different containers with clear labels. UV-treated seawater with an irradiation intensity of 1,000 mWh/L was injected, and the seawater temperature was 22°C; the UV-treated seawater was changed every 60 min until gamete release. Sperm-egg contact was strictly prevented in the operation process.
[31] 3) The impurities were filtered and removed from the sperms and eggs using a 150 m screen, respectively; and the oocyte quality and sperm motility were determined through microscopic examination to ensure that there were no fertilized eggs in the eggs; the eggs and sperm determined by microscopic examination were placed in 50 L and 10 L plastic boxes for later use, respectively.
[32] 4) The spare sperms and eggs were mixed; through microscopic examination, the ratio of sperm to egg was (15-20):1; subsequently, the fertilized eggs were divided into three groups and labeled as DF, DDF-CB, and DDF-6-DMAP.
[33] 5) The development of the fertilized eggs in the control group was observed at intervals through microscopic examination. When the proportion of PB1 was 70%, the fertilized eggs in the DDF-CB and DDF-6-DMAP groups were treated in 1.50 mg/L CB L for 15 min or 30 mg/L 6-DMAP for 15 min, respectively.
[34] 6) After timing was over, the fertilized eggs were washed twice in1% DMSO for 15 min each time.
[35] 7) Allotriploid larvae were obtained according to conventional hatching and post breeding methods. Veliger larvae (24 h) were taken and the ploidy was determined by flow cytometry. The triploid induction rates of the DDF-CB and DDF-6-DMAP groups were 84.12+6.71% and 100.00+0.00%, respectively.
[36] 8) The seawater temperature was 20.0-21.5°C during fertilization and veliger larval hatching.
[37] Example 2
[38] 1) H. discus hannai and H.fulgens with excellent individual vigor and without external damage were selected as broodstocks; the H. discus hannai (Y) had an average shell length of 72.943.31 mm, a shell width of 48.733.50 mm, and a wet weight of 50.93+9.10 g; the H. fulgens (o) had an average shell length of 75.93+0.98 mm, a shell width of 51.31±1.36 mm, and a wet weight of 52.223.50 g. The broodstocks were bred in different parent abalone culture ponds, respectively, and the sex characteristics of parent abalones in each parent abalone culture pond must be single.
[39] 2) After the gonadal maturation, the broodstocks were dried in the shade for 60 min. Subsequently, male and female individuals were placed in different containers with clear labels. UV-treated seawater with an irradiation intensity of 900 mWh/L was injected, and the sea water temperature was 22°C; the UV-treated seawater was changed every 60 min until gamete release. Sperm-egg contact was strictly prevented in the operation process.
[40] 3) The impurities were filtered and removed from the sperms and eggs using a 150 m screen, respectively; and the oocyte quality and sperm motility were determined through microscopic examination to ensure that there were no fertilized eggs in the eggs; the eggs and sperm determined by microscopic examination were placed in 50 L and 10 L plastic boxes for later use, respectively.
[41] 4) The spare sperms and eggs were mixed; through microscopic examination, the ratio of sperm to egg was (10-7):1; subsequently, the fertilized eggs were divided into three groups and labeled as DF, DDF-CB, and DDF-6-DMAP.
[42] 5) The development of the fertilized eggs in the control group was observed at intervals through microscopic examination. When the proportion of PB1 was 80%, the fertilized eggs in the DDF-CB and DDF-6-DMAP groups were rapidly treated in 1.50 mg/L CB for 15 min and mg/L 6-DMAP L for 15 min, respectively.
[43] 6) After timing was over, the fertilized eggs were washed twice in1% DMSO for 15 min each time.
[44] 7) Allotriploid larvae were obtained according to conventional hatching and post breeding methods. Veliger larvae (24 h) were taken and the ploidy was determined by flow cytometry. The triploid induction rates of the DDF-CB and DDF-6-DMAP groups were 84.52+7.15% and 100.00+0.00%, respectively.
[45] 8) The seawater temperature was 20.1-21.6°C during fertilization and veliger larval hatching.
[46] Example 3
[47] 1) H. discus hannai and H.fulgens with excellent individual vigor and without external damage were selected as broodstocks; the H. discus hannai (Y) had an average shell length of 76.615.06 mm, a shell width of 51.952.88 mm, and a wet weight of 54.43+7.57 g; the H. fulgens (o) had an average shell length of 71.700.96 mm, a shell width of 49.51+2.76 mm, and a wet weight of 51.664.62 g. The broodstocks were bred in different parent abalone culture ponds, respectively, and the sex characteristics of parent abalones in each parent abalone culture pond must be single.
[48] 2) After the gonadal maturation, the broodstocks were dried in the shade for 60 min. Subsequently, male and female individuals were placed in different containers with clear labels. UV-treated seawater with an irradiation intensity of 1,000 mWh/L was injected, and the sea water temperature was 21°C; the UV-treated seawater was changed every 60 min until gamete release. Sperm-egg contact was strictly prevented in the operation process.
[49] 3) The impurities were filtered and removed from the sperms and eggs using a 150 m screen, respectively; and the oocyte quality and sperm motility were determined through microscopic examination to ensure that there were no fertilized eggs in the eggs; the eggs and sperm determined by microscopic examination were placed in 50 L and 10 L plastic boxes for later use, respectively.
[50] 4) The spare sperms and eggs were mixed; through microscopic examination, the ratio of sperm to egg was (15-20):1; subsequently, the fertilized eggs were divided into three groups and labeled as DF, DDF-CB, and DDF-6-DMAP.
[51] 5) The development of the fertilized eggs in the control group was observed at intervals through microscopic examination. When the proportion of PB1 was 50%, the fertilized eggs in the DDD-CB and DDD-6-DMAP groups were rapidly treated in 1.75 mg/L CB for 20 min and mg/L 6-DMAP L for 10 min, respectively.
[52] 6) After timing was over, the fertilized eggs were washed twice in1% DMSO for 15 min each time.
[53] 7) H. discus hannaitriploid larvae were obtained according to conventional hatching and post-breeding methods. Veliger larvae (24 h) were taken and the ploidy was determined by flow cytometry. The triploid induction rates of the DDF-CB and DDF-6-DMAP groups were 100.00+0.00% and 100.00+0.00%, respectively.
[54] 8) The seawater temperature was 21.5-22.5°C during fertilization and veliger larval hatching.
Claims (5)
1. A method comprising the steps of: step 1, broodstock collection and conditioning; step 2, artificial induction; step 3, selection and treatment of gametes; step 4, drug induction; and step 5, drug removal.
2. The method for chemically inducing an allotriploid abalone according to claim 1, wherein in step 1, the broodstock collection and conditioning is specifically implemented by: selecting Haliotis discus hannai (Y) and Haliotis fulgens (o) with excellent individual vigor and no external damage as broodstocks, placing the abalones in parent abalone culture ponds for accelerated maturation, respectively; the broodstocks are Haliotis discus hannaiaged 2-3 years, and have a shell length of greater than 70 mm and a body weight of greater than 50 g; sex characteristics in the parent abalone culture ponds must be single, and seawater temperature in the abalone culture ponds is 24-26°C.
3. The method for chemically inducing an allotriploid abalone according to claim 1, wherein in step 2, the artificial induction is specifically implemented by: using a method of exposing to air for desiccation stimulation combined with ultraviolet (UV) irradiation of seawater to induce individuals with gonadal maturation until broodstock pawn and release sperms.
4. The method for chemically inducing an allotriploid abalone according to claim 3, wherein the exposing to air for desiccation stimulation is implemented by: after exposing to air for 60 120 min, strictly distinguishing between male and female individuals into different containers with clear labels, injecting UV-treated seawater with an irradiation intensity of 500-1,200 mWh/L UV, changing the seawater every 60 min until gamete release, and taking care to prevent unintended fertilization.
5. The method for chemically inducing an allotriploid abalone according to claim 1, wherein in step 3, the selection and treatment of gametes is specifically implemented by: filtering and removing impurities from the gametes in step 2 using a 150 m screen, and determining oocyte quality and sperm motility through microscopic examination; the microscopic examination includes that in an egg fluid with high quality of the microscopic examination, an egg has an intact egg membrane and a regular shape without unintended fertilization, and a sperm with excellent vigor has excellent mobility and dispersion; wherein in step 4, the drug induction is specifically implemented by: observing the development of fertilized eggs in the control group, and quickly adding the fertilized eggs in the treatment group into a 1.50-1.75 mg/L CB induction buffer to treat for 15-20 min or a 30-40 mg/L 6-dimethylaminopurine (6-DMAP) induction buffer to treat for 10-15 min when 50%-80% of first polar bodies appear; wherein the first polar bodies should be sampled and observed microscopically at intervals during the observation; the CB induction buffer is obtained by diluting a 1 mg/mL stock solution prepared by dissolving CB in dimethyl sulfoxide (DMSO), and the 6-DMAP induction buffer is obtained from a 50 mg/mL stock solution prepared by dissolving 6-DMAP in DMSO; and wherein in step 5, the drug removal is implemented by: placing drug-treated fertilized eggs in 1% DMSO and washing twice, 15 min each time..
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