CN107142239A - The method for improving ox IVF Embryos culture efficiency - Google Patents
The method for improving ox IVF Embryos culture efficiency Download PDFInfo
- Publication number
- CN107142239A CN107142239A CN201710433656.6A CN201710433656A CN107142239A CN 107142239 A CN107142239 A CN 107142239A CN 201710433656 A CN201710433656 A CN 201710433656A CN 107142239 A CN107142239 A CN 107142239A
- Authority
- CN
- China
- Prior art keywords
- nutrient solution
- amino acid
- ivf embryos
- concentration
- embryo
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 210000002257 embryonic structure Anatomy 0.000 title claims abstract description 83
- 238000000034 method Methods 0.000 title claims abstract description 49
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 claims abstract description 113
- 235000015097 nutrients Nutrition 0.000 claims abstract description 102
- 229960003180 glutathione Drugs 0.000 claims abstract description 55
- 238000000338 in vitro Methods 0.000 claims abstract description 50
- 150000001413 amino acids Chemical class 0.000 claims abstract description 41
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims abstract description 36
- DAEPDZWVDSPTHF-UHFFFAOYSA-M sodium pyruvate Chemical compound [Na+].CC(=O)C([O-])=O DAEPDZWVDSPTHF-UHFFFAOYSA-M 0.000 claims abstract description 36
- 108010024636 Glutathione Proteins 0.000 claims abstract description 27
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 26
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims abstract description 22
- 230000000694 effects Effects 0.000 claims abstract description 20
- 239000003797 essential amino acid Substances 0.000 claims abstract description 20
- 235000020776 essential amino acid Nutrition 0.000 claims abstract description 20
- 239000001103 potassium chloride Substances 0.000 claims abstract description 18
- 239000011780 sodium chloride Substances 0.000 claims abstract description 18
- 229940054269 sodium pyruvate Drugs 0.000 claims abstract description 18
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 claims abstract description 16
- RGHNJXZEOKUKBD-MGCNEYSASA-N D-galactonic acid Chemical compound OC[C@@H](O)[C@H](O)[C@H](O)[C@@H](O)C(O)=O RGHNJXZEOKUKBD-MGCNEYSASA-N 0.000 claims abstract description 16
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims abstract description 16
- 239000011575 calcium Substances 0.000 claims abstract description 16
- 229910052791 calcium Inorganic materials 0.000 claims abstract description 16
- 239000008103 glucose Substances 0.000 claims abstract description 15
- 235000017557 sodium bicarbonate Nutrition 0.000 claims abstract description 11
- 229910000030 sodium bicarbonate Inorganic materials 0.000 claims abstract description 11
- 239000000243 solution Substances 0.000 claims description 103
- 210000001161 mammalian embryo Anatomy 0.000 claims description 59
- 229940024606 amino acid Drugs 0.000 claims description 40
- 235000001014 amino acid Nutrition 0.000 claims description 40
- 210000004027 cell Anatomy 0.000 claims description 38
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 claims description 35
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 claims description 35
- 239000001509 sodium citrate Substances 0.000 claims description 34
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 claims description 34
- 210000002459 blastocyst Anatomy 0.000 claims description 33
- 230000012447 hatching Effects 0.000 claims description 31
- 230000004720 fertilization Effects 0.000 claims description 30
- 239000007788 liquid Substances 0.000 claims description 26
- 230000013020 embryo development Effects 0.000 claims description 25
- GUBGYTABKSRVRQ-QUYVBRFLSA-N beta-maltose Chemical compound OC[C@H]1O[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@@H]1O GUBGYTABKSRVRQ-QUYVBRFLSA-N 0.000 claims description 23
- 239000007864 aqueous solution Substances 0.000 claims description 16
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 claims description 15
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 12
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 claims description 12
- 239000002904 solvent Substances 0.000 claims description 12
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 claims description 10
- 210000004681 ovum Anatomy 0.000 claims description 10
- 102000002322 Egg Proteins Human genes 0.000 claims description 8
- 108010000912 Egg Proteins Proteins 0.000 claims description 8
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 claims description 7
- HHGZUQPEIHGQST-RGVONZFCSA-N (2r)-2-amino-3-[[(2r)-2-amino-2-carboxyethyl]disulfanyl]propanoic acid;dihydrochloride Chemical compound Cl.Cl.OC(=O)[C@@H](N)CSSC[C@H](N)C(O)=O HHGZUQPEIHGQST-RGVONZFCSA-N 0.000 claims description 6
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 6
- 239000004471 Glycine Substances 0.000 claims description 6
- FFEARJCKVFRZRR-UHFFFAOYSA-N L-Methionine Natural products CSCCC(N)C(O)=O FFEARJCKVFRZRR-UHFFFAOYSA-N 0.000 claims description 6
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 claims description 6
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 claims description 6
- BVHLGVCQOALMSV-JEDNCBNOSA-N L-lysine hydrochloride Chemical compound Cl.NCCCC[C@H](N)C(O)=O BVHLGVCQOALMSV-JEDNCBNOSA-N 0.000 claims description 6
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 claims description 6
- 229930195722 L-methionine Natural products 0.000 claims description 6
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 claims description 6
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 claims description 6
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 claims description 6
- 239000004473 Threonine Substances 0.000 claims description 6
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 claims description 6
- 235000004279 alanine Nutrition 0.000 claims description 6
- 239000007789 gas Substances 0.000 claims description 6
- 229960004452 methionine Drugs 0.000 claims description 6
- 229960002898 threonine Drugs 0.000 claims description 6
- 239000004474 valine Substances 0.000 claims description 6
- ODHCTXKNWHHXJC-VKHMYHEASA-N 5-oxo-L-proline Chemical compound OC(=O)[C@@H]1CCC(=O)N1 ODHCTXKNWHHXJC-VKHMYHEASA-N 0.000 claims description 5
- CKLJMWTZIZZHCS-UHFFFAOYSA-N D-OH-Asp Natural products OC(=O)C(N)CC(O)=O CKLJMWTZIZZHCS-UHFFFAOYSA-N 0.000 claims description 5
- CKLJMWTZIZZHCS-UWTATZPHSA-N L-Aspartic acid Natural products OC(=O)[C@H](N)CC(O)=O CKLJMWTZIZZHCS-UWTATZPHSA-N 0.000 claims description 5
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 claims description 5
- 229960005261 aspartic acid Drugs 0.000 claims description 5
- 229940098773 bovine serum albumin Drugs 0.000 claims description 5
- 229960005190 phenylalanine Drugs 0.000 claims description 5
- 210000000130 stem cell Anatomy 0.000 claims description 5
- 238000012549 training Methods 0.000 claims description 5
- 108700026215 vpr Genes Proteins 0.000 claims description 5
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 claims description 4
- 210000001771 cumulus cell Anatomy 0.000 claims description 4
- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 claims description 3
- DWNBOPVKNPVNQG-LURJTMIESA-N (2s)-4-hydroxy-2-(propylamino)butanoic acid Chemical compound CCCN[C@H](C(O)=O)CCO DWNBOPVKNPVNQG-LURJTMIESA-N 0.000 claims description 3
- QAWLKTDBUQOFEF-UHFFFAOYSA-N 3-(4-bromophenyl)propanenitrile Chemical compound BrC1=CC=C(CCC#N)C=C1 QAWLKTDBUQOFEF-UHFFFAOYSA-N 0.000 claims description 3
- 102000029816 Collagenase Human genes 0.000 claims description 3
- 108060005980 Collagenase Proteins 0.000 claims description 3
- 108010003272 Hyaluronate lyase Proteins 0.000 claims description 3
- 102000001974 Hyaluronidases Human genes 0.000 claims description 3
- 229960002424 collagenase Drugs 0.000 claims description 3
- 230000029087 digestion Effects 0.000 claims description 3
- 229920002674 hyaluronan Polymers 0.000 claims description 3
- 229960003160 hyaluronic acid Drugs 0.000 claims description 3
- 229960002773 hyaluronidase Drugs 0.000 claims description 3
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 claims 6
- 241000432824 Asparagus densiflorus Species 0.000 claims 1
- 229960002989 glutamic acid Drugs 0.000 claims 1
- 229960002885 histidine Drugs 0.000 claims 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 claims 1
- -1 KH2PO3 Chemical compound 0.000 abstract description 4
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 abstract description 4
- 229910001629 magnesium chloride Inorganic materials 0.000 abstract description 2
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 abstract 2
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 abstract 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 abstract 1
- 238000005516 engineering process Methods 0.000 description 27
- 238000007792 addition Methods 0.000 description 24
- 238000002474 experimental method Methods 0.000 description 24
- 241000283690 Bos taurus Species 0.000 description 16
- 230000006907 apoptotic process Effects 0.000 description 14
- 238000004519 manufacturing process Methods 0.000 description 10
- 230000035800 maturation Effects 0.000 description 10
- 239000013589 supplement Substances 0.000 description 10
- 241001465754 Metazoa Species 0.000 description 8
- 210000000472 morula Anatomy 0.000 description 8
- 229920000089 Cyclic olefin copolymer Polymers 0.000 description 7
- 238000003776 cleavage reaction Methods 0.000 description 7
- 238000010790 dilution Methods 0.000 description 7
- 239000012895 dilution Substances 0.000 description 7
- 230000007017 scission Effects 0.000 description 7
- 210000000582 semen Anatomy 0.000 description 7
- 230000008859 change Effects 0.000 description 6
- 238000011161 development Methods 0.000 description 6
- 230000018109 developmental process Effects 0.000 description 6
- 239000000203 mixture Substances 0.000 description 6
- 210000000287 oocyte Anatomy 0.000 description 6
- 235000008708 Morus alba Nutrition 0.000 description 5
- 238000009395 breeding Methods 0.000 description 5
- 230000001488 breeding effect Effects 0.000 description 5
- 230000006872 improvement Effects 0.000 description 5
- 238000011160 research Methods 0.000 description 5
- 240000000249 Morus alba Species 0.000 description 4
- 229930182555 Penicillin Natural products 0.000 description 4
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 4
- 239000012091 fetal bovine serum Substances 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 238000012986 modification Methods 0.000 description 4
- 230000004048 modification Effects 0.000 description 4
- 239000001301 oxygen Substances 0.000 description 4
- 229910052760 oxygen Inorganic materials 0.000 description 4
- 229940049954 penicillin Drugs 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 108010083123 CDX2 Transcription Factor Proteins 0.000 description 3
- 102000006277 CDX2 Transcription Factor Human genes 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 3
- 241000124008 Mammalia Species 0.000 description 3
- 241000283973 Oryctolagus cuniculus Species 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 210000001109 blastomere Anatomy 0.000 description 3
- 238000012214 genetic breeding Methods 0.000 description 3
- VEXZGXHMUGYJMC-UHFFFAOYSA-N hydrochloric acid Substances Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 3
- 244000144972 livestock Species 0.000 description 3
- 230000003647 oxidation Effects 0.000 description 3
- 238000007254 oxidation reaction Methods 0.000 description 3
- 230000009467 reduction Effects 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- QZNNVYOVQUKYSC-JEDNCBNOSA-N (2s)-2-amino-3-(1h-imidazol-5-yl)propanoic acid;hydron;chloride Chemical class Cl.OC(=O)[C@@H](N)CC1=CN=CN1 QZNNVYOVQUKYSC-JEDNCBNOSA-N 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- 101100298998 Caenorhabditis elegans pbs-3 gene Proteins 0.000 description 2
- 102000003952 Caspase 3 Human genes 0.000 description 2
- 108090000397 Caspase 3 Proteins 0.000 description 2
- LEVWYRKDKASIDU-QWWZWVQMSA-N D-cystine Chemical compound OC(=O)[C@H](N)CSSC[C@@H](N)C(O)=O LEVWYRKDKASIDU-QWWZWVQMSA-N 0.000 description 2
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 2
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 2
- 150000008536 L-asparagines Chemical class 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 239000003963 antioxidant agent Substances 0.000 description 2
- 230000003078 antioxidant effect Effects 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- RYYVLZVUVIJVGH-UHFFFAOYSA-N caffeine Chemical compound CN1C(=O)N(C)C(=O)C2=C1N=CN2C RYYVLZVUVIJVGH-UHFFFAOYSA-N 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 235000018417 cysteine Nutrition 0.000 description 2
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 2
- 229960003067 cystine Drugs 0.000 description 2
- 238000004043 dyeing Methods 0.000 description 2
- 229920000669 heparin Polymers 0.000 description 2
- 229960002897 heparin Drugs 0.000 description 2
- 238000007689 inspection Methods 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 238000005457 optimization Methods 0.000 description 2
- 210000001672 ovary Anatomy 0.000 description 2
- 210000003101 oviduct Anatomy 0.000 description 2
- 239000002504 physiological saline solution Substances 0.000 description 2
- 229920000136 polysorbate Polymers 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- VOXZDWNPVJITMN-ZBRFXRBCSA-N 17β-estradiol Chemical compound OC1=CC=C2[C@H]3CC[C@](C)([C@H](CC4)O)[C@@H]4[C@@H]3CCC2=C1 VOXZDWNPVJITMN-ZBRFXRBCSA-N 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- OQUFOZNPBIIJTN-UHFFFAOYSA-N 2-hydroxypropane-1,2,3-tricarboxylic acid;sodium Chemical compound [Na].OC(=O)CC(O)(C(O)=O)CC(O)=O OQUFOZNPBIIJTN-UHFFFAOYSA-N 0.000 description 1
- 235000006810 Caesalpinia ciliata Nutrition 0.000 description 1
- 241000059739 Caesalpinia ciliata Species 0.000 description 1
- 206010067477 Cytogenetic abnormality Diseases 0.000 description 1
- 230000005778 DNA damage Effects 0.000 description 1
- 231100000277 DNA damage Toxicity 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- LPHGQDQBBGAPDZ-UHFFFAOYSA-N Isocaffeine Natural products CN1C(=O)N(C)C(=O)C2=C1N(C)C=N2 LPHGQDQBBGAPDZ-UHFFFAOYSA-N 0.000 description 1
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 1
- 241000218231 Moraceae Species 0.000 description 1
- 244000303589 Morus indica Species 0.000 description 1
- 241000337007 Oceania Species 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- DYUQAZSOFZSPHD-UHFFFAOYSA-N Phenylpropyl alcohol Natural products CCC(O)C1=CC=CC=C1 DYUQAZSOFZSPHD-UHFFFAOYSA-N 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 238000000540 analysis of variance Methods 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000003975 animal breeding Methods 0.000 description 1
- 230000001640 apoptogenic effect Effects 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 238000005842 biochemical reaction Methods 0.000 description 1
- 230000008033 biological extinction Effects 0.000 description 1
- 230000029803 blastocyst development Effects 0.000 description 1
- 229960001948 caffeine Drugs 0.000 description 1
- VJEONQKOZGKCAK-UHFFFAOYSA-N caffeine Natural products CN1C(=O)N(C)C(=O)C2=C1C=CN2C VJEONQKOZGKCAK-UHFFFAOYSA-N 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 230000005754 cellular signaling Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 238000005056 compaction Methods 0.000 description 1
- 238000010835 comparative analysis Methods 0.000 description 1
- 238000000280 densification Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000008143 early embryonic development Effects 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 229960005309 estradiol Drugs 0.000 description 1
- 229930182833 estradiol Natural products 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 210000001733 follicular fluid Anatomy 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 230000014509 gene expression Effects 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- QWPPOHNGKGFGJK-UHFFFAOYSA-N hypochlorous acid Chemical compound ClO QWPPOHNGKGFGJK-UHFFFAOYSA-N 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000036512 infertility Effects 0.000 description 1
- 208000000509 infertility Diseases 0.000 description 1
- 231100000535 infertility Toxicity 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- JEIPFZHSYJVQDO-UHFFFAOYSA-N iron(III) oxide Inorganic materials O=[Fe]O[Fe]=O JEIPFZHSYJVQDO-UHFFFAOYSA-N 0.000 description 1
- 230000006651 lactation Effects 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 239000002480 mineral oil Substances 0.000 description 1
- 235000010446 mineral oil Nutrition 0.000 description 1
- 238000011017 operating method Methods 0.000 description 1
- 210000002394 ovarian follicle Anatomy 0.000 description 1
- 230000004792 oxidative damage Effects 0.000 description 1
- 230000010627 oxidative phosphorylation Effects 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 230000002572 peristaltic effect Effects 0.000 description 1
- 230000008823 permeabilization Effects 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 230000035935 pregnancy Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 150000003254 radicals Chemical class 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 239000003642 reactive oxygen metabolite Substances 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000001568 sexual effect Effects 0.000 description 1
- 230000008010 sperm capacitation Effects 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000002277 temperature effect Effects 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000002054 transplantation Methods 0.000 description 1
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0603—Embryonic cells ; Embryoid bodies
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/05—Inorganic components
- C12N2500/10—Metals; Metal chelators
- C12N2500/12—Light metals, i.e. alkali, alkaline earth, Be, Al, Mg
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/05—Inorganic components
- C12N2500/10—Metals; Metal chelators
- C12N2500/12—Light metals, i.e. alkali, alkaline earth, Be, Al, Mg
- C12N2500/16—Magnesium; Mg chelators
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/32—Amino acids
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/34—Sugars
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/999—Small molecules not provided for elsewhere
Abstract
The present invention relates to the method for improving ox IVF Embryos culture efficiency.Specifically, one aspect of the present invention is related to a kind of ox IVF Embryos nutrient solution, and the nutrient solution is included：NaCl, KCl, NaHCO3, MgCl2, KH2PO3, Sodium Pyruvate, glucose, galactonic acid calcium, hyclone, L glutamine, essential amino acid, nonessential amino acid, glutathione and water.Further relate in the method for the efficiency culture ox IVF Embryos of raising, this method comprises the following steps：Ox IVF Embryos are placed in ox IVF Embryos nutrient solution, In vitro culture is carried out under 38.5 DEG C, 0.5%CO2,100% damp condition.Technique effect excellent as used in the description is presented in the inventive method and the nutrient solution used.
Description
Technical field
The invention belongs to technical field of animal reproduction, it is related to a kind of technology for agricultural-animal and veterinary breeding, specifically relates to
And a kind of method for improving ox IVF Embryos culture efficiency, more particularly, it relates to which ox IVF Embryos are trained
Application of the nutrient solution in ox IVF Embryos culture.Further, the invention further relates to trained using the ox IVF Embryos
The method that nutrient solution carries out ox IVF Embryos culture.Particularly, the method for ox IVF Embryos culture of the present invention has excellent
Different cost advantage.
Background technology
(In Vitro Fertilization) or (external fertilization) in vitro fertilization refer to that lactation is moved
The technology of fertilization process is completed in the environment that the sperm and ovum of thing are manually controlled in vitro, English abbreviation is IVF.Due to it with
Embryo transfer technology (ET) is inseparable, and referred to as IVF-ET.In biology, after IVF to parent
The animal of acquisition claims test tube animal (test-tube animal).The success of this technology is in the 1950s, at nearest 20 years
Quickly grow, reached its maturity and turned into an important and conventional animal reproduction biotechnology.
Technology in vitro fertilization has important to animals' reproduction study mechanism, husbandry sector, medical science and animals on the brink of extinction protection etc.
Meaning.Such as make experiment material with mouse, rat or rabbit, technology in vitro fertilization can be used for research mammalian gamete to occur,
Fertilization and early embryo development mechanism.In livestock animal breeding, technology in vitro fertilization provides cheap and high for Embryo Production
The means of effect, to making full use of improved seeds resource, shorten the livestock reproduction cycle, and accelerating breed improvement speed etc. has important valency
Value.In the mankind, IVF-ET technologies are some infertilities for the treatment of and overcome one of important measures of sex-kink disease.Technology in vitro fertilization
Or the indispensable parts of modern biotechnology such as mammal embryo transplanting, clone, transgenosis and sexual control.
With the development of modern agriculture science and technology, in order to more make full use of the breeding potential of breeding cow, accelerate genetic breeding
Process, turns into inevitable in production practices using efficient breeding new technology.Ovum pick-up (Ovum pick UP, OPU) and body
Outer fertilization technique (In Vitro Fertilization, IVF) is embryo's work that 1980s fast development is got up
Journey new technology, the two, which is combined, can obtain the clear and definite embryo of a large amount of Genetic lineages, so as to shorten the generation inteval.At present, both
It is important that technology has become the farmer of the animal husbandry developed countries such as American-European and Oceania to expand that breeding cow group uses
Reproduction technique.However, using conventional ox Embryo Culture system (CR1aa and SOF liquid), ox blastocyst rate in vitro fertilization compared with
It is low, and embryo quality causes the pregnancy rate after embryo transplantation acceptor low, therefore how to improve blastaea also far away from internal embryo
Developmental rate and embryo quality turn into the emphasis that IVF Embryos are produced and the focus studied.
Early in 1878, German Scnenk just using rabbit and cavy as material, start to explore mammal it is external by
Smart technology.But until nineteen fifty-one, Chinese American Zhang Minjue and Austin have found after sperm microcytotoxicity phenomenon respectively, in vitro fertilization
Technology just obtains breakthrough.Ox technology in vitro fertilization by the maturation in vitro of egg mother cell, the In-vitro Capacitation of sperm, by
The influence of many aspects such as the vitro culture conditions of smart ovum.
The in vitro culture of embryo is a key link of IVF technologies, is also oocyte in vitro maturation and in vitro fertilization
The embodiment and inspection of technology final effect.After fertilization in vitro, embryonated egg need to undergo a series of into blastaea growth course
Important change, includes formation, First cleavage, the activation of embryonic gene group, densification and the formation blastaea of zygote.This
During, the change of external environment can cause gene expression to change, so as to influence normal development and the quality of embryo.Mesh
Before, the in vitro culture research of Mammalian Embryo is concentrated mainly on improvement nutrient solution composition to meet different developmental phases
Embryotrophy demand.Based on (Rosenkrans, C.F., Jr.and N.L.First, Effect of free such as Rosenkrans
amino acids and vitamins on cleavage and developmental rate of bovine zygotes
In vitro.J Anim Sci, 1994.72 (2):P.434-7) Charles Rosenkrans 1 (CR1) nutrient solution of exploitation
With (Tervit, H.R., D.G.Whittingham, and L.E.Rowson, Successful the culture in such as Tervit
vitro of sheep and cattle ova.J Reprod Fertil,1972.30(3):P.493-7) synthesis of exploitation is defeated
Oviduct liquid (Synthetic Oviductal Fluid, SOF), two kinds of cultivating systems have been gradually formed through updating for many years.According to
(Sagirkaya, H., et al., Developmental potential of bovine such as Hakan Sagirkaya
oocytes cultured in different maturation and culture conditions.Anim Reprod
Sci,2007.101(3-4):) and (Somfai, T., et al., Development of bovine such as Somfai p.225-40
embryos cultured in CR1aa and IVD101media using different oxygen tensions and
culture systems.Acta Vet Hung,2010.58(4):P.465-74) achievement in research shows, CR1aa nutrient solutions are used
There is preferable effect in ox Embryo Culture, can be widely applied to the Embryo Culture of ox；Thompson, J.G. etc. (Thompson,
J.G.,et al.,Effect of inhibitors and uncouplers of oxidative phosphorylation
during compaction and blastulation of bovine embryos cultured in vitro.J
Reprod Fertil,2000.118(1):) and (Feugang, J.M., the O.Camargo- such as Jean M.Feugang p.47-55
Rodriguez,and E.Memili,Culture systems for bovine embryos.Livestock Science,
2009.121(2-3):P.141-149 result of study) shows that SOF nutrient solutions are also a kind of training for being suitable for ox Embryo Culture
The system of supporting.Zhang Zhi's equality (Zhang Zhiping, An Zhixing, rust, Zhang Yong, the optimization Xibei Univ. of Agricultural ＆ Forest Science ＆ Technology of ox Embryo Culture system
Journal, 2006.34) and Sang Guojun etc. (Sang Guojun, bovine oocyte and external embryo culture technique research .2008) result of study
It has been shown that, CR1aa the and SOF nutrient solutions by optimization are adapted to the embryo in vitro culture of ox, obtain good culture effect.
Mammalian Embryo development is the process of a hight coordinate and accurate adjustment.During evolution, gametid is progressively
A series of molecule cascade network is formd, to ensure that embryonic development period is systematically carried out.In growth course, embryo's is interior
Outer active oxygen radical (Reactive Oxygen Species, ROS) and the balance of antioxidant are played to early embryonic development
Decisive role.
Most biochemical reaction produces ROS, its in the cell, it is outer play the role of important, a part of ROS plays signal
The effect of molecule, but most of ROS are harmful to body.(Brooker, R.J., the Genetics such as Brooker, R.J.:
Analysis and principles (4th ed.) .McGraw-Hill Science, 2011) report, ROS can cause carefully
Born of the same parents' DNA damage, the oxidation of unrighted acid, the oxidation of Amino Acids in Proteins even can cause the inactivation of some enzymes.One
As for, ROS exists in four kinds of forms, and wherein H2O2 oxidations are stronger, is the main factor for causing oxidative damage.
Numerous studies show that glutathione (GSH) is a kind of antioxidant existed in nonprotein form, can be removed
A variety of free radicals：Ultra-oxygen anion free radical, hydroxyl radical free radical, hydrogen peroxide, hypochlorous acid and fat oxygen radical, and can tie up
Hold intraor extracellular redox equilibrium.Intraor extracellular environment GSH and ROS level is to influence two weights during development of fertilized ova
Want factor.Early in, (de Matos, D.G.and C.C.Furnus, the The importance of such as de Matos in 2000
having high glutathione(GSH)level after bovine in vitro maturation on embryo
development effect of beta-mercaptoethanol,cysteine and
cystine.Theriogenology,2000.53(3):P.761-71) once by adding β-mercapto during Embryo Culture in vitro
Base ethanol, cysteine plus cystine improve blastocyst rate.
Although technology in vitro fertilization can be successfully applied to many mammals, because blastocyst rate in vitro fertilization is low and lead
Cause production cost height, the efficiency of IVF Embryos low, limit extensive use of the technology in the practice of ox fast-propagation.Cause
This, how reducing cost and improving ox IVF Embryo Productions efficiency and embryo quality turns into urgent problem to be solved.
At present, it is main using CR1aa and SOF liquid as embryo in-vitro culture solution in ox technical system in vitro fertilization, and herein
On the basis of be improved, blastocyst rate has different degrees of raising, blastocyst rate average out to 30%-40%.For capsule
Idioplasm amount, can be assessed by blastomere sum, ICM cell numbers/total cell number ratio, apoptosis rate.Blastomere
Different and different, (Iwasaki, the S.and T.Nakahara, Cell such as S.Iwasaki in sum stage according to residing for blastaea
number and incidence of chromosomal anomalies in bovine blastocysts
fertilized in vitro followed by culture in vitro or in vivo in rabbit
oviducts.Theriogenology,1990.33(3):P.669-75) the ox early blastocyst total cell number average out to 44 obtained,
ICM cell numbers/blastaea total cell number ratio is 15.8% or so；Andrew J.Watson etc. (Watson, A.J., et al.,
Impact of bovine oocyte maturation media on oocyte transcript levels,
blastocyst development,cell number,and apoptosis.Biol Reprod,2000.62(2):
P.355-64) statistics ox blastomere apoptosis rate is about 7.7%-13%.
CN103898046B (Chinese Patent Application No. 201410073635.4) discloses one kind, and to be exclusively used in ox in vitro fertilization
The nutrient solution of embryo, the culture formula of liquid is：NaCl 109.5mM、KCl 3.1mM、NaHCO3 26.2mM、MgCl2·
6H2O 0.8mM, KH2PO31.19mM, Sodium Pyruvate 0.4mM, glucose 1.5mM, galactonic acid calcium 5mM, 10v/v% tire ox blood
Clearly, Glu 1mM, 2v/v% essential amino acids, 1v/v% nonessential amino acids and glutathione 3mM, are prepared with water；Institute
The aqueous solution prepared after essential amino acid is mixed in proportion for following amino acid is stated, wherein, each amino acid content is：L- hydrochloric acid essence
Propylhomoserin 6.32g/L, the water thing 2.1g/L of CYSTINE dihydrochloride 1.564g/L, L- histidine monohydrochloride one, ILE
2.625g/L, L-Leu 2.62g/L, L lysine HCL 3.625g/L, L-Methionine 0.755g/L, L-phenylalanine
1.65g/L, L-threonine 2.38g/L, L-Trp 0.51g/L, TYR 1.8g/L and Valine 2.34g/L；It is described
Nonessential amino acid is the aqueous solution prepared after following amino acid is mixed in proportion, wherein, each amino acid content is：ALANINE
The water thing 1.5g/L of 0.89g/L, L- asparagine one, L-Aspartic acid 1.33g/L, Pidolidone 1.47g/L, glycine
0.75g/L, L-PROLINE 1.15g/L and Serine 1.05g/L.Ox IVF Embryos are placed in above-mentioned nutrient solution and carried out
In vitro culture, it is believed that result, which is shown, is substantially better than the control group for being not added with GSH, improves blastocyst rate and embryo quality, drop
The cost of low produced in vitro embryo, is applied to practice for ox IVF technologies and provides experiment basis, can greatly accelerate that genetic breeding enters
Journey.
Other bibliography：
Old big first fertilizations biology .2003, Beijing:Science Press；
Fang Junshun bovine embryo vitros production technical research《The agriculture sections of Chinese excellent MA theses full-text database
Skill volume》.2007, (the 4th phase)；
The influence of the clear cumulus cells of Cao Hai and condition of culture to ox embryo production in vitro efficiency《The outstanding master's degree of China
Paper full-text database agricultural science and technologys volume》.2008, (the 9th phase)；
I.H.KIM et al.Effect of exogenous glutathione on the in vitro
fertilization of bovine oocytes.Theriogenology.1999,Vol.52(3)。
However, the method for having the culture of the improved ox IVF Embryos of performance is still expected in this area, such as still phase
The method of ox IVF Embryos culture efficiency to be improved, for example, have the ox IVF Embryos with excellent cost advantage
The method of culture.
The content of the invention
It is an object of the invention to provide a kind of method of the culture of the ox IVF Embryos of performance improvement, particularly phase
Ox IVF Embryo Productions efficiency to be improved and embryo quality.More specifically, the present invention needs to provide a kind of to reach above-mentioned purpose
It is exclusively used in the nutrient solution of ox IVF Embryos and and using the method for culture progress ox IVF Embryos culture.This hair
A person of good sense has had now surprisingly been found that, excellent technique effect is presented using the inventive method, the present invention is consequently found that and be able to
Complete.
Therefore, first aspect present invention provides a kind of ox IVF Embryos nutrient solution, the nutrient solution is included：
109-110mM NaCl,
2.9-3.1mM KCl,
26.0-26.5mM NaHCO3,
0.5-1.0mM MgCl26H2O,
1.0-1.3mM KH2PO3,
0.4mM Sodium Pyruvate,
1.5mM glucose,
5mM galactonic acid calcium,
2-3v/v% hyclone,
1mM Glu,
2v/v% essential amino acid,
1v/v% nonessential amino acid,
1-10mM glutathione and
It is used as the water with liquid solvent.
The ox IVF Embryos nutrient solution of any embodiment according to a first aspect of the present invention, the nutrient solution is included：
109mM NaCl,
3.1mM KCl,
26.2mM NaHCO3,
0.8mM MgCl26H2O,
1.19mM KH2PO3,
0.4mM Sodium Pyruvate,
1.5mM glucose,
5mM galactonic acid calcium,
2.5v/v% hyclone,
1mM Glu,
2v/v% essential amino acid,
1v/v% nonessential amino acid,
1-10mM glutathione and
It is used as the water with liquid solvent.
The ox IVF Embryos nutrient solution of any embodiment according to a first aspect of the present invention, wherein the required amino
Acid is as the aqueous solution obtained by following amino acid is prepared with concentration in proportion：
L- R-genes 6.32g/L,
CYSTINE dihydrochloride 1.564g/L,
The water thing 2.1g/L of L- histidine monohydrochlorides one,
ILE 2.625g/L,
L-Leu 2.62g/L,
L lysine HCL 3.625g/L,
L-Methionine 0.755g/L,
L-phenylalanine 1.65g/L,
L-threonine 2.38g/L,
L-Trp 0.51g/L,
TYR 1.8g/L and
Valine 2.34g/L.
The ox IVF Embryos nutrient solution of any embodiment according to a first aspect of the present invention, wherein the nonessential ammonia
Base acid is as the aqueous solution obtained by following amino acid is prepared with concentration in proportion：
ALANINE 0.89g/L,
The water thing 1.5g/L of L- asparagines one,
L-Aspartic acid 1.33g/L,
Pidolidone 1.47g/L,
Glycine 0.75g/L,
L-PROLINE 1.15g/L and
Serine 1.05g/L.
The ox IVF Embryos nutrient solution of any embodiment according to a first aspect of the present invention, wherein in the nutrient solution
The concentration of glutathione is 1~7mM.
The ox IVF Embryos nutrient solution of any embodiment according to a first aspect of the present invention, wherein in the nutrient solution
The concentration of glutathione is 3~5mM.
The ox IVF Embryos nutrient solution of any embodiment according to a first aspect of the present invention, wherein in the nutrient solution
The concentration of glutathione is 1mM, 3mM, 5mM or 7mM.
The ox IVF Embryos nutrient solution of any embodiment according to a first aspect of the present invention, wherein in the nutrient solution
The concentration of glutathione is 3mM or 5mM.
The ox IVF Embryos nutrient solution of any embodiment according to a first aspect of the present invention, wherein in the nutrient solution
The concentration of glutathione is 3mM.
The ox IVF Embryos nutrient solution of any embodiment according to a first aspect of the present invention, wherein also including citric acid
Sodium and maltose.In one embodiment, in the ox IVF Embryos nutrient solution sodium citrate and maltose concentration
Respectively 0.03~0.05w/v% and 0.01~0.03w/v%.In one embodiment, the ox IVF Embryos training
The concentration of sodium citrate and maltose is respectively 0.04w/v% and 0.02w/v% in nutrient solution.It has been had now surprisingly been found that,
When adding micro sodium citrate and maltose simultaneously in ox IVF Embryos nutrient solution, hatching rate is favorably improved,
And other indexs are unaffected.Hatching rate is the important evaluation index of quality of blastocysts, impregnable in other indexs
Under the premise of improve hatching rate, it is significant for ox IVF Embryos training.Still further, it was discovered that, adding
Plus in the case of this sodium citrate and maltose, the dense of hyclone in ox IVF Embryos nutrient solution can be greatly reduced
Degree, and obtain essentially identical culture effect；Due in ox IVF Embryos nutrient solution hyclone relative to therein
Other components (including sodium citrate and maltose) are prohibitively expensive, and the prime cost of ox IVF Embryos nutrient solution is
Hyclone, it is extremely that the consumption of hyclone, which reduces for improving ox IVF Embryos culture efficiency, reduction production cost,
Favourable.
Further, second aspect of the present invention provides a kind of side of efficiency culture ox IVF Embryos with raising
Method, this method comprises the following steps：
Ox IVF Embryos are placed in ox IVF Embryos nutrient solution, 38.5 DEG C, it is 0.5%CO2,100% wet
In vitro culture is carried out under the conditions of degree.
The method of any embodiment according to a second aspect of the present invention, wherein In vitro culture comprises the following steps：
(1) smart ovum is incubated after 8h altogether, takes out egg mother cell, using 1mg/ml hyaluronic acid collagenase treatment 3min, so
Blown and beaten repeatedly using liquid-transfering gun afterwards, until cumulus cell comes off substantially；
(2) digestion of hyaluronidase is terminated using the TCM199 containing 10%FBS, is being added doubtful embryonated egg with mouth suction pipe
Washed in embryo's early stage nutrient solution added with bovine serum albumin(BSA) (being generally also abbreviated as BSA) after 3 times, be put into embryo's early stage nutrient solution
Cultivated in drop, condition of culture is 38.5 DEG C, 0.5%CO2,100% humidity；
(3) after 48h, the spilting of an egg to the embryo of 4-8 cells is moved into late embryogenesis nutrient solution, and (it is that ox of the invention is external
Fertilized embryo nutrient solution) drop in cultivated (condition of culture be 38.5 DEG C, 0.5%CO2,100% humidity), the later half amounts of 48h are more
Change late embryogenesis nutrient solution and continue to cultivate (38.5 DEG C, 5%CO2 gases, 95% humidity)；
(4) timing was started for 0 day with the same day of being fertilized, counts blastocyst rate during to the 7th day, hatching is counted during to the 9th day
Rate (it is percentage obtained by hatched blastocyst number divided by blastaea number).
Included in the method for any embodiment according to a second aspect of the present invention, the ox IVF Embryos nutrient solution：
109-110mM NaCl,
2.9-3.1mM KCl,
26.0-26.5mM NaHCO3,
0.5-1.0mM MgCl26H2O,
1.0-1.3mM KH2PO3,
0.4mM Sodium Pyruvate,
1.5mM glucose,
5mM galactonic acid calcium,
2-3v/v% hyclone,
1mM Glu,
2v/v% essential amino acid,
1v/v% nonessential amino acid,
1-10mM glutathione and
It is used as the water with liquid solvent.
Included in the method for any embodiment according to a second aspect of the present invention, the ox IVF Embryos nutrient solution：
109mM NaCl,
3.1mM KCl,
26.2mM NaHCO3,
0.8mM MgCl26H2O,
1.19mM KH2PO3,
0.4mM Sodium Pyruvate,
1.5mM glucose,
5mM galactonic acid calcium,
2.5v/v% hyclone,
1mM Glu,
2v/v% essential amino acid,
1v/v% nonessential amino acid,
1-10mM glutathione and
It is used as the water with liquid solvent.
It is described in the method for any embodiment according to a second aspect of the present invention, the ox IVF Embryos nutrient solution
Essential amino acid is as the aqueous solution obtained by following amino acid is prepared with concentration in proportion：
L- R-genes 6.32g/L,
CYSTINE dihydrochloride 1.564g/L,
The water thing 2.1g/L of L- histidine monohydrochlorides one,
ILE 2.625g/L,
L-Leu 2.62g/L,
L lysine HCL 3.625g/L,
L-Methionine 0.755g/L,
L-phenylalanine 1.65g/L,
L-threonine 2.38g/L,
L-Trp 0.51g/L,
TYR 1.8g/L and
Valine 2.34g/L.
It is described in the method for any embodiment according to a second aspect of the present invention, the ox IVF Embryos nutrient solution
Nonessential amino acid is as the aqueous solution obtained by following amino acid is prepared with concentration in proportion：
ALANINE 0.89g/L,
The water thing 1.5g/L of L- asparagines one,
L-Aspartic acid 1.33g/L,
Pidolidone 1.47g/L,
Glycine 0.75g/L,
L-PROLINE 1.15g/L and
Serine 1.05g/L.
The method of any embodiment according to a second aspect of the present invention, wherein the concentration of the nutrient solution GSH-PX activity is
1~7mM.
The method of any embodiment according to a second aspect of the present invention, wherein the concentration of the nutrient solution GSH-PX activity is
3~5mM.
The method of any embodiment according to a second aspect of the present invention, wherein the concentration of the nutrient solution GSH-PX activity is
1mM, 3mM, 5mM or 7mM.
The method of any embodiment according to a second aspect of the present invention, wherein the concentration of the nutrient solution GSH-PX activity is
3mM or 5mM.
The method of any embodiment according to a second aspect of the present invention, wherein the concentration of the nutrient solution GSH-PX activity is
3mM。
Also included in the method for any embodiment according to a second aspect of the present invention, the ox IVF Embryos nutrient solution
Sodium citrate and maltose.In one embodiment, sodium citrate and maltose in the ox IVF Embryos nutrient solution
Concentration be respectively 0.03~0.05w/v% and 0.01~0.03w/v%.In one embodiment, the ox is in vitro fertilization
The concentration of sodium citrate and maltose is respectively 0.04w/v% and 0.02w/v% in embryo medium.
Any technical characteristic that any embodiment of either side or the either side of the present invention has is equally applicable
Any embodiment of other any embodiments or other either sides, as long as they will not be conflicting, certainly mutual
Between where applicable, if necessary can individual features be made with appropriate modification.Make to various aspects of the present invention with feature into one below
The description of step.
All documents recited in the present invention, their full content is incorporated herein by reference, and if these are literary
Offer expressed implication with it is of the invention inconsistent when, be defined by the statement of the present invention.In addition, the various terms that use of the present invention and
Phrase has well known to a person skilled in the art general sense, nonetheless, the present invention remain desirable at this to these terms and
Phrase is described in more detail and explained that the term and phrase referred to is if any inconsistent with common art-recognized meanings, with institute's table of the present invention
The implication stated is defined.
The hyclone that the present invention is used readily can buy its from market and standardize commercial form, for example can be from each
Plant Australia hyclone (article No. that Gibco companies are bought at agent：10099141), New Zealand's hyclone (article No.：
10091148), North America hyclone (article No.：16000044), Mexico's hyclone (article No.：10437028) etc., these business
The price 500ml of product hyclone is most more than 8000 yuan, tire during in ox IVF Embryos nutrient solution of the present invention
Cow's serum is cost significant contributor.In the experiment of the context of the invention, if not otherwise specified, hyclone used is
Australia hyclone (article No. of Gibco companies：10099141).
The present invention has advantages below：The present invention in nutrient solution by adding certain density glutathione
(Glutathione, GSH), can significantly improve the developmental potency of embryo in vitro.Differential dyeing is carried out to blastaea simultaneously, distinguishes interior
Cell mass (Inner Cell Mass, ICM), trophocyte and apoptotic cell, compare total cell number, and calculate ICM cells
Number/total cell number ratio and apoptosis rate, as a result show addition GSH test group be better than control group, improve blastocyst rate and
Embryo quality, reduces the cost of produced in vitro embryo, and being applied to practice for ox IVF technologies provides experiment basis, can greatly accelerate
Genetic breeding process.In addition, having had now surprisingly been found that, the ox IVF Embryos nutrient solution with inventive formulation is used
Raising hatching rate can be significantly improved, and other indexs are unaffected, and hatching rate is that the important of quality of blastocysts is commented
Valency index, improves hatching rate on the premise of other indexs are unaffected, has for the training of ox IVF Embryos
It is significant.In addition, it has also been found that, in the case where adding this sodium citrate and maltose, ox can be greatly reduced
The concentration of hyclone in IVF Embryos nutrient solution, and obtain essentially identical culture effect；Because ox is in vitro fertilization
Hyclone is prohibitively expensive, ox relative to other components (including sodium citrate and maltose) therein in embryo medium
The prime cost of IVF Embryos nutrient solution is hyclone, and the consumption reduction of hyclone is in vitro fertilization for improving ox
Embryo Culture efficiency, reduction production cost are extremely advantageous.
Embodiment
The present invention can be further described by the following examples, however, the scope of the present invention is not limited
In following embodiments.One of skill in the art, can be with it is understood that on the premise of without departing substantially from the spirit and scope of the present invention
Various change and modification are carried out to the present invention.The present invention carries out general to the material and test method that are arrived used in experiment
And/or specific description.Although for realize many materials used in the object of the invention and operating method be it is known in the art that
But the present invention is still described in detail as far as possible herein.Unless otherwise specified, technological means used in embodiment is ability
Conventional meanses known to field technique personnel, raw materials used is commercial goods.
Embodiment 1：The cultural method of ox IVF Embryos
First, reagent
Maturation culture solution：The LH of FSH, 10IU/ml containing 0.01IU/ml, 1 μ g/ml estradiol, 100ng/mlIGF,
50ng/ml EGF, 100U/ml penicillin, 100 μ g/ml streptomysins, the TCM-199 nutrient solutions of 10% hyclone.
Wash seminal fluid：KCl, 2.25mM of NaCl, 4.02mM containing 112.0mM CaCl22H2O, 0.52mM's
Heparin, the 4mg/ of MgCl26H2O, 0.83mM KH2PO3,37.0mM NaHCO3,1.25mM Sodium Pyruvate, 10 μ g/ml
Ml bovine serum albumin(BSA) (Bovine serum albumin, BSA), 10mM caffeine, 100U/ml penicillin, 100 μ
The aqueous solution of g/ml streptomysin.
By seminal fluid：KCl, 2.25mM of NaCl, 4.02mM containing 112.0mM CaCl22H2O, 0.52mM's
Heparin, the 4mg/ of MgCl26H2O, 0.83mM KH2PO3,37.0mM NaHCO3,1.25mM Sodium Pyruvate, 10 μ g/ml
The aqueous solution of ml BSA, 100U/ml penicillin, 100 μ g/ml streptomysin.
Embryo's early stage nutrient solution：KCl, 26.2mM of NaCl, 3.1mM containing 109.5mM NaHCO3,0.8mM's
MgCl26H2O, 1.19mM KH2PO3,0.4mM Sodium Pyruvate, 1.5mM glucose, 5mM galactonic acid calcium, 6mg/
Ml BSA, 1mM Glu, 2v/v% essential amino acids, the aqueous solution of 1v/v% nonessential amino acids.
Late embryogenesis nutrient solution：KCl, 26.2mM of NaCl, 3.1mM containing 109.5mM NaHCO3,0.8mM's
MgCl26H2O, 1.19mM KH2PO3,0.4mM Sodium Pyruvate, 1.5mM glucose, 5mM galactonic acid calcium, 10v/
V% hyclones (Fetal bovine serum, FBS), 1mM Glu, 2v/v% essential amino acids, 1v/v% are non-
The aqueous solution of necessary amino acid.
The essential amino acid be following amino acid in proportion/concentration prepare the aqueous solution, wherein, each amino acid content
For：L- R-genes 6.32g/L, CYSTINE dihydrochloride 1.564g/L, L- histidine monohydrochloride one water thing 2.1g/L, L- are different
Leucine 2.625g/L, L-Leu 2.62g/L, L lysine HCL 3.625g/L, L-Methionine 0.755g/L, L- phenylpropyl alcohol
Propylhomoserin 1.65g/L, L-threonine 2.38g/L, L-Trp 0.51g/L, TYR 1.8g/L and Valine 2.34g/L.
The nonessential amino acid be following amino acid in proportion/concentration prepare the aqueous solution, wherein, each amino acid content
For：The water thing 1.5g/L of ALANINE 0.89g/L, L- asparagine one, L-Aspartic acid 1.33g/L, Pidolidone 1.47g/
L, glycine 0.75g/L, L-PROLINE 1.15g/L and Serine 1.05g/L.
2nd, the collection of egg mother cell and maturation in vitro
1. gathering ox ovary from slaughterhouse, it is placed in 37 DEG C of physiological saline, laboratory is delivered in 3h.
2. ovary is cleaned 3-5 times using the physiological saline of the warm containing penicillin and streptomysin.
3. using vacuum peristaltic pump, the liquor folliculi in 5-8 millimeters of ovarian follicles is extracted with No. 18 syringe needles.
4. selecting oocyte-cumulus cell conjugate (COCs) under stereomicroscope, COCs is washed in egg-cleaning liquid
Twice, it washed once in maturation culture solution.
5. the COCs of wash clean to be put into containing maturation culture solution and cover in four orifice plates of mineral oil, 50 pieces are cultivated per hole
COCs, cultivates 22-24h.
3rd, it is in vitro fertilization
1. COCs is moved into 50 μ l fertilization drops from maturation culture solution, often drop puts 15 pieces of COCs.
2.38 DEG C of water-bath defrosting seminal fluid, wash seminal fluid using 5ml and 5min are centrifuged under the conditions of 500g；Supernatant is abandoned, 5ml is added
Seminal fluid centrifugation 5min is washed, supernatant is abandoned, using seminal fluid adjustment sperm concentration is washed, it is about 2 × 10 to make sperm concentration7Individual/ml.
3. taking 50 μ l seminal fluid, 100 μ l drop is mixed into the fertilization drop containing COCs.
4.38.5 DEG C, smart ovum is incubated 8h altogether under 5%CO2 gases, 95% damp condition.
4th, In vitro culture
1. smart ovum is incubated after 8h altogether, egg mother cell is taken out, using 1mg/ml hyaluronic acid collagenase treatment 3min, then
Blown and beaten repeatedly using liquid-transfering gun, until cumulus cell comes off substantially.
2. terminating the digestion of hyaluronidase using the TCM199 containing 10%FBS, doubtful embryonated egg is being added with mouth suction pipe
Washed in embryo's early stage nutrient solution added with bovine serum albumin(BSA) after 3 times, be put into embryo's early stage culture drop and cultivated (38.5
DEG C, 5%CO2 gases, 95% humidity).
After 3.48h, the spilting of an egg to the embryo of 4-8 cells is moved to cultivated in late embryogenesis culture drop (38.5 DEG C,
5%CO2 gases, 95% humidity), the later half amounts of 48h change late embryogenesis nutrient solution and continue culture (38.5 DEG C, 5%CO2 gases,
95% humidity).
4. the same day started timing for 0 day to be fertilized, blastocyst rate is counted during to the 7th day, to statistics hatching rate at the 9th day
(it is percentage obtained by hatched blastocyst number divided by blastaea number).
5th, the most suitable addition concentration screenings of GSH (glutathione) in late embryogenesis nutrient solution
1. the GSH for preparing 0.5M is used as liquid storage.
2. using GSH liquid storages, add and match somebody with somebody into late embryogenesis nutrient solution according to final concentration of 1mM, 3mM, 5mM, 7mM gradient
The test group of late embryogenesis nutrient solution is made as, using non-addition group as control.
3. 48h respectively at culture, the 5th day, the 7th day, counts the cleavage egg and 8 cell rates, mulberry body rate of each group
And blastocyst rate.
6th, embryo's differential dyeing
1. choosing the in vitro culture blastaea of the 7th day, 20min is fixed using 2% paraformaldehyde.
2. washed twice using the phosphate buffer (PBS-BSA) containing 0.5%BSA, be put into permeabilization liquid (50 μ l Triton,
5 μ l Tween 80s and 9.945ml PBS) in, room temperature places 30min.
3. using 2M hydrochloric acid room temperature treatment 20min, then using 100mM Tris-HCl room temperature treatment 10min, make CDX2
Albumen can be with an anti-binding.
4. being cleaned three times using PBS-BSA, blastaea is put into confining liquid (1ml lowlenthal serums, 5 μ l Tween 80s and 8.995ml
PBS in), then room temperature closing 1h is transferred to 4 DEG C of refrigerator closings and stays overnight.
5. discarding confining liquid, CDX2 primary antibodies press 1 with confining liquid:200 dilutions, are incubated at room temperature 2h, discard primary antibody dilution, use
PBS-BSA is cleaned 3 times, each 5min.
6.caspase-3 primary antibodies (being purchased from Cell Signaling Technology companies) press 1 with confining liquid:200 is dilute
Release, be incubated at room temperature 2h, discard primary antibody dilution, cleaned with PBS-BSA 3 times, each 5min.
7. press 1 with confining liquid under the conditions of lucifuge:The 200 specific secondary antibodies of dilution CDX2 (being purchased from Sigma companies), at room temperature
Avoid light place 1h.Secondary antibody dilution is discarded under the conditions of lucifuge, with PBS 3 times, each 5min.
8. press 1 with confining liquid under the conditions of lucifuge:The 200 specific secondary antibodies of dilution caspase-3 (are purchased from Life
Technologies companies), avoid light place 1h at room temperature.Secondary antibody dilution is discarded under the conditions of lucifuge, with PBS 3 times, every time
5min。
9. add the 10 μ g/mL dye liquors of Hochest 33342 dye nucleus, room temperature effect 5min, under fluorescence microscope
Observe and take pictures.
10. experiment is in triplicate, 10 blastaeas are randomly selected every time, are calculated apoptosis rate, ICM cell numbers/total cell number and are come
Assess quality of blastocysts.
7th, data statistics
Experimental data uses the ANOVA programs in statistical software SAS V8 to be analyzed, Duncan ' s multiple-
The significance of difference between range method of inspection determination processings, works as p<Significant difference is thought when 0.05.
8th, the developmental rate that GSH is remarkably improved bovine IVF embryo is added in late embryogenesis nutrient solution
Selection GSH adds concentration and screened for 0,1,3,5,7mM, by counting cleavage rates (that is, cleavage rates=embryonated egg
Split number/embryonated egg number), 4-8 cell rates, mulberry body rate, blastocyst rate (that is, blastocyst rate=blastaea number/spilting of an egg embryo number).
As a result：
In late embryogenesis nutrient solution GSH add concentration be 0,1,3,5, the cleavage rates between five groups of 7mM and 4-8 cell rates it is equal
Not notable (the p of difference>0.05), such as five groups cleavage rates are in the range of 0.80~0.88, five groups of 4-8 cell rates 0.67~
In the range of 0.79；
To morula developmental rate (i.e. mulberry body rate, morula rate=mulberries embryo number/spilting of an egg embryo number), 3mM and 5mM's
GSH addition groups are all remarkably higher than control group and 5mM, 7mM experimental group (p<, but this two group difference of 3mM and 5mM is not notable 0.05)
(p>0.05), this three groups mulberry body rate of such as control group and 5mM, 7mM is in the range of 0.46~0.49, and 3mM and 5mM this two
The mulberry body rate of group is in the range of 0.61~0.63；
For blastocyst rate, 1mM, 3mM and 5mM GSH addition groups are all remarkably higher than control group (p<0.05), and 7mM
GSH additions group is with compareing group difference not significantly (p>0.05), such as control group and the blastocyst rate of 7mM groups are respectively 0.32 He
The blastocyst rate of 0.36,1mM group is 0.42,3mM and the blastocyst rate of 5mM groups is in the range of 0.46~0.51；These results with
The result of CN103898046A tables 1 is basically identical.It can be seen that GSH is added in late embryogenesis nutrient solution can improve ox IVF embryos'
Development rate.
9th, the quality of ox IVF Embryos can be improved by GSH being added in late embryogenesis nutrient solution
By counting total cell number, ICM cell numbers/total cell number, apoptosis rate (that is, apoptosis rate=apoptosis cell/total thin
Born of the same parents number), the quality of blastocysts that comparative analysis GSH difference addition concentration groups are obtained.As a result it is as follows：
For total cell number, tetra- GSH concentration addition groups of 1mM, 3mM, 5mM, 7mM are all remarkably higher than control group (p<
, and 3mM, 5mM and 7mM group are all remarkably higher than 1mM groups (p 0.05)<0.05), such as 0mM groups total cell number is that 42.4,1mM groups are total
Cell number is 63.6, and 3mM, 5mM and 7mM group total cell number are in the range of 82~89；
For the ratio of ICM cell numbers/total cell number, 1mM additions group and the not notable (p of control group difference>0.05), 3mM,
5mM and 7mM groups are substantially less than control group (p<0.05) difference not significantly (p, but between this three groups>0.05), such as 1mM is added
ICM cell numbers/total cell number ratio with control group is organized in the range of 0.41~0.48, the ICM cells of 3mM, 5mM and 7mM group
Number/total cell number is than in the range of 0.27~0.34 in the range of 0.41~0.48；
For apoptosis rate, the not notable (p of difference between four addition groups and control group>0.05), 1mM, 3mM, 7mM GSH
The apoptosis rate of addition group and control group is substantially less than 5mM addition groups (p<0.05)；These results and the knot of CN103898046A tables 2
It is really basically identical.It can be seen that GSH addition is favorably improved the quality of ox IVF Embryos.
Tenth, the most suitable addition concentration that GSH is added in late embryogenesis nutrient solution is 3mM
In four experimental groups, the morula rate and blastocyst rate that 3mM and 5mM GSH additions group is obtained are all remarkably higher than pair
According to group and other experimental group (p<0.05)；But the morula rate of 3mM GSH addition groups and 5mM GSH addition group differences do not show
Write (p>, but blastocyst rate is significantly higher than 5mM addition groups (p 0.05)<0.05), it is seen that the ox IVF embryos in 3mM GSH addition groups
The developmental rate of tire is highest, therefore GSH optimum concentration is 3mM GSH addition groups.
The present embodiment has filtered out most suitable addition concentration, drastically increases blastaea production efficiency, and blastocyst rate is carried by 30%
Up to 50%；Embryo quality is estimated and displayed that, quality of blastocysts is also superior to control group after addition this product.
Embodiment 2：The cultural method of ox IVF Embryos
With reference to embodiment 1, different is only that late embryogenesis nutrient solution composition therein is changed to：
109mM NaCl,
3.1mM KCl,
26.0mM NaHCO3,
0.5mM MgCl26H2O,
1.3mM KH2PO3,
0.4mM Sodium Pyruvate,
1.5mM glucose,
5mM galactonic acid calcium,
10v/v% hyclone,
1mM Glu,
2v/v% essential amino acid,
1v/v% nonessential amino acid,
3mM glutathione and
It is used as the water with liquid solvent.
Tested according to embodiment 1, as a result glutathione group basic phase of the indices with using 3mM in embodiment 1
Together, for example morula rate is that 0.633, blastocyst rate is 0.508, apoptosis rate 0.050 in the present embodiment.
Embodiment 3：The cultural method of ox IVF Embryos
With reference to embodiment 1, different is only that late embryogenesis nutrient solution composition therein is changed to：
110mM NaCl,
2.9mM KCl,
26.5mM NaHCO3,
1.0mM MgCl26H2O,
1.0mM KH2PO3,
0.4mM Sodium Pyruvate,
1.5mM glucose,
5mM galactonic acid calcium,
10v/v% hyclone,
1mM Glu,
2v/v% essential amino acid,
1v/v% nonessential amino acid,
3mM glutathione and
It is used as the water with liquid solvent.
Tested according to embodiment 1, as a result glutathione group basic phase of the indices with using 3mM in embodiment 1
Together, for example morula rate is that 0.636, blastocyst rate is 0.521, apoptosis rate 0.050 in the present embodiment.
Embodiment 4：The cultural method of ox IVF Embryos
With reference to embodiment 1, different is only that late embryogenesis nutrient solution composition therein is changed to：
109mM NaCl,
3.1mM KCl,
26.0mM NaHCO3,
0.5mM MgCl26H2O,
1.3mM KH2PO3,
0.4mM Sodium Pyruvate,
1.5mM glucose,
5mM galactonic acid calcium,
10v/v% hyclone,
1mM Glu,
2v/v% essential amino acid,
1v/v% nonessential amino acid,
3mM glutathione,
Sodium citrate 0.04w/v%,
Maltose 0.02w/v% and
It is used as the water with liquid solvent.
As a result show, in terms of the indices that embodiment 1 is investigated, the present embodiment 4 is with using 3mM's in embodiment 1
Glutathione group is essentially identical, such as morula rate is that 0.634, blastocyst rate is 0.518, apoptosis rate 0.049 in the present embodiment 4；
In addition, counting hatching rate at the 9th day, as a result show the present embodiment hatching rate up to 71.6%；Other statistics hair
Existing, embodiment 1~3 is all using hatching rate in the experiment of 3mM glutathione in the range of 36~39%.
In the experiment of a supplement, with reference to above-described embodiment 4, different is only that sodium citrate concentration is changed to 0.03w/
V% or 0.05w/v% (but maltose is still 0.02w/v%), in two experiments indices with the basic phase of above-described embodiment 4
Together, such as hatching rate is up to 70.2~71.4% scopes.It is different with reference to above-described embodiment 4 in the experiment of a supplement
Only it is that maltose concentration is changed to 0.01w/v% or 0.03w/v% (but sodium citrate is still 0.04w/v%), it is each in two experiments
Index is essentially identical with above-described embodiment 4, and such as hatching rate is up in the range of 71.6~72.6%；This result table
It is bright, it can effectively improve hatching rate after the appropriate sodium citrate of supplement addition and maltose in late embryogenesis nutrient solution.
In the experiment of a supplement, with reference to above-described embodiment 4, different is only that (but maltose is still without sodium citrate
0.02w/v%), or it is different be only without maltose (but sodium citrate is still 0.04w/v%), in two experiments except
Indices beyond hatching rate are essentially identical with above-described embodiment 4, but hatching rate is significantly small, 38
(p in the range of~40%<0.05, with significant difference)；This result shows, only add both sodium citrate and maltose it
Hatching rate can not be effectively improved for the moment.
Embodiment 5：The cultural method of ox IVF Embryos
With reference to embodiment 1, different is only that late embryogenesis nutrient solution composition therein is changed to：
109mM NaCl,
3.1mM KCl,
26.0mM NaHCO3,
0.5mM MgCl26H2O,
1.3mM KH2PO3,
0.4mM Sodium Pyruvate,
1.5mM glucose,
5mM galactonic acid calcium,
2.5v/v% hyclone,
1mM Glu,
2v/v% essential amino acid,
1v/v% nonessential amino acid,
3mM glutathione,
Sodium citrate 0.04w/v%,
Maltose 0.02w/v% and
It is used as the water with liquid solvent.
As a result show, in terms of the indices that embodiment 1 is investigated, the present embodiment 5 is with using 3mM's in embodiment 1
Glutathione group is essentially identical, such as morula rate is that 0.628, blastocyst rate is 0.522, apoptosis rate 0.048 in the present embodiment 5；
In addition, counting hatching rate at the 9th day, as a result show the present embodiment hatching rate up to 70.2%；Other statistics hair
Existing, embodiment 1~3 is all using hatching rate in the experiment of 3mM glutathione in the range of 36~39%.
In the experiment of a supplement, with reference to above-described embodiment 5, different is only that sodium citrate concentration is changed to 0.03w/
V% or 0.05w/v% (but maltose is still 0.02w/v%), in two experiments indices with the basic phase of above-described embodiment 5
Together, such as hatching rate is up to 69.7~71.8% scopes.It is different with reference to above-described embodiment 5 in the experiment of a supplement
Only it is that maltose concentration is changed to 0.01w/v% or 0.03w/v% (but sodium citrate is still 0.04w/v%), it is each in two experiments
Index is essentially identical with above-described embodiment 5, and such as hatching rate is up in the range of 71.8~72.4%；This result table
It is bright, it can effectively improve hatching rate after the appropriate sodium citrate of supplement addition and maltose in late embryogenesis nutrient solution.
In the experiment of a supplement, with reference to above-described embodiment 5, different is only that (but maltose is still without sodium citrate
0.02w/v%), or it is different be only without maltose (but sodium citrate is still 0.04w/v%), in two experiments except
Indices beyond hatching rate are essentially identical with above-described embodiment 5, but hatching rate is significantly small, 36
(p in the range of~39%<0.05, with significant difference)；This result shows, only add both sodium citrate and maltose it
Hatching rate can not be effectively improved for the moment.In the experiment of a supplement, with reference to above-described embodiment 5, different is only not add
Plus sodium citrate and maltose, the indices in experiment in addition to hatching rate are essentially identical with above-described embodiment 5,
It is 31.3% (p but hatching rate is significantly small<0.05, with significant difference)；This result shows, by tire ox
If blastaea can not be effectively improved during without both sodium citrate and maltose in the case that serum-concentration is down to 2.5v/v%
Incubation rate.In the experiment of a supplement, with reference to above-described embodiment 5, different be only hyclone concentration be changed to 2v/v% or
3v/v%, indices are essentially identical with above-described embodiment 5 in two experiments, and such as hatching rate is up to 70.3~71.7%
In the range of；This result shows, is supplemented in late embryogenesis nutrient solution in the case of adding appropriate sodium citrate and maltose, i.e.,
Make to remain able to effectively improve hatching rate when hyclone concentration is down into 2~3v/v% scope, now tire ox blood
The consumption of clear this expensive reagent is the 20~30% of the consumption of embodiment 1, and the cost of increased sodium citrate and maltose
It is low-down.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (10)
1. a kind of ox IVF Embryos nutrient solution, the nutrient solution is included：109-110mM NaCl, 2.9-3.1mM KCl,
26.0-26.5mM NaHCO3,0.5-1.0mM MgCl26H2O, 1.0-1.3mM KH2PO3,0.4mM Sodium Pyruvate,
1.5mM glucose, 5mM galactonic acid calcium, 2-3v/v% hyclone, 1mM Glu, 2v/v% it is required
Amino acid, 1v/v% nonessential amino acid, 1-10mM glutathione and be used as match somebody with somebody liquid solvent water.
2. ox IVF Embryos nutrient solution according to claim 1, the nutrient solution is included：109mM NaCl, 3.1mM's
KCl, 26.2mM NaHCO3,0.8mM MgCl26H2O, 1.19mM KH2PO3,0.4mM Sodium Pyruvate, 1.5mM
Glucose, 5mM galactonic acid calcium, 2.5v/v% hyclone, 1mM Glu, 2v/v% essential amino acid,
1v/v% nonessential amino acid, 1-10mM glutathione and be used as match somebody with somebody liquid solvent water.
3. ox IVF Embryos nutrient solution according to claim 1, wherein the essential amino acid be by following amino acid by
The aqueous solution obtained by ratio and concentration preparation：L- R-genes 6.32g/L, CYSTINE dihydrochloride 1.564g/L, L- salt
The sour water thing 2.1g/L of histidine one, ILE 2.625g/L, L-Leu 2.62g/L, L lysine HCL 3.625g/
L, L-Methionine 0.755g/L, L-phenylalanine 1.65g/L, L-threonine 2.38g/L, L-Trp 0.51g/L, TYR
1.8g/L and Valine 2.34g/L.
4. ox IVF Embryos nutrient solution according to claim 1, wherein the nonessential amino acid is by following amino acid
The aqueous solution obtained by being prepared in proportion with concentration：Water thing 1.5g/L, L- asparagus fern of ALANINE 0.89g/L, L- asparagine one
Propylhomoserin 1.33g/L, Pidolidone 1.47g/L, glycine 0.75g/L, L-PROLINE 1.15g/L and Serine 1.05g/L.
5. ox IVF Embryos nutrient solution according to claim 1, wherein the concentration of the nutrient solution GSH-PX activity be 1~
7mM；For example, 3~5mM；For example, 1mM, 3mM, 5mM or 7mM；For example, 3mM or 5mM；For example, 3mM.
6. ox IVF Embryos nutrient solution according to claim 1, wherein also including sodium citrate and maltose；For example, institute
The concentration for stating sodium citrate and maltose in ox IVF Embryos nutrient solution be respectively 0.03~0.05w/v% and 0.01~
0.03w/v%；For example, the concentration of sodium citrate and maltose is respectively 0.04w/ in the ox IVF Embryos nutrient solution
V% and 0.02w/v%.
7. in the method for the efficiency culture ox IVF Embryos of raising, this method comprises the following steps：By ox embryo in vitro fertilization
Tire is placed in ox IVF Embryos nutrient solution, and embryo is carried out under 38.5 DEG C, 0.5%CO2,100% damp condition and is trained in vitro
Support.
8. method according to claim 7, wherein In vitro culture comprise the following steps：
(1) smart ovum is incubated after 8h altogether, is taken out egg mother cell, using 1mg/ml hyaluronic acid collagenase treatment 3min, is then made
Blown and beaten repeatedly with liquid-transfering gun, until cumulus cell comes off substantially；
(2) using containing 10%FBS TCM199 terminate hyaluronidase digestion, with mouth suction pipe by doubtful embryonated egg added with
Wash after 3 times, be put into embryo's early stage culture drop in embryo's early stage nutrient solution of bovine serum albumin(BSA) (being generally also abbreviated as BSA)
Cultivated, condition of culture is 38.5 DEG C, 0.5%CO2,100% humidity；
(3) after 48h, the spilting of an egg to the embryo of 4-8 cells is moved into late embryogenesis nutrient solution, and (it is that ox of the invention is in vitro fertilization
Embryo medium) (condition of culture is 38.5 DEG C, 0.5%CO2,100% humidity) is cultivated in drop, 48h later half amounts replacing embryo
Tire later stage nutrient solution simultaneously continues to cultivate (38.5 DEG C, 5%CO2 gases, 95% humidity)；
(4) timing was started for 0 day with the same day of being fertilized, blastocyst rate is counted during to the 7th day, to statistics hatching rate at the 9th day.
9. method according to claim 7, wherein：
Included in the ox IVF Embryos nutrient solution：109-110mM NaCl, 2.9-3.1mM KCl, 26.0-26.5mM
NaHCO3,0.5-1.0mM MgCl26H2O, 1.0-1.3mM KH2PO3,0.4mM Sodium Pyruvate, 1.5mM grape
Sugar, 5mM galactonic acid calcium, 2-3v/v% hyclone, 1mM Glu, 2v/v% essential amino acid, 1v/
V% nonessential amino acid, 1-10mM glutathione and be used as match somebody with somebody liquid solvent water；Or
Included in the ox IVF Embryos nutrient solution：109mM NaCl, 3.1mM KCl, 26.2mM NaHCO3,
0.8mM MgCl26H2O, 1.19mM KH2PO3,0.4mM Sodium Pyruvate, 1.5mM glucose, 5mM galactonic acid
Calcium, 2.5v/v% hyclone, 1mM Glu, 2v/v% essential amino acid, 1v/v% nonessential amino
Acid, 1-10mM glutathione and as match somebody with somebody liquid solvent water.
10. method according to claim 7, wherein：
The essential amino acid in the ox IVF Embryos nutrient solution is prepared in proportion with concentration by following amino acid
The aqueous solution of gained：L- R-genes 6.32g/L, the water thing of CYSTINE dihydrochloride 1.564g/L, L- histidine monohydrochloride one
2.1g/L, ILE 2.625g/L, L-Leu 2.62g/L, L lysine HCL 3.625g/L, L-Methionine
0.755g/L, L-phenylalanine 1.65g/L, L-threonine 2.38g/L, L-Trp 0.51g/L, TYR 1.8g/L and
Valine 2.34g/L；
The nonessential amino acid in the ox IVF Embryos nutrient solution is matched somebody with somebody in proportion with concentration by following amino acid
The aqueous solution obtained by system：The water thing 1.5g/L of ALANINE 0.89g/L, L- asparagine one, L-Aspartic acid 1.33g/L, L-
Glutamic acid 1.47g/L, glycine 0.75g/L, L-PROLINE 1.15g/L and Serine 1.05g/L；
Sodium citrate and maltose are also included in the ox IVF Embryos nutrient solution, such as described ox IVF Embryos training
The concentration of sodium citrate and maltose is respectively 0.03~0.05w/v% and 0.01~0.03w/v%, such as described ox in nutrient solution
The concentration of sodium citrate and maltose is respectively 0.04w/v% and 0.02w/v% in IVF Embryos nutrient solution；And/or
The concentration of the nutrient solution GSH-PX activity is 1~7mM, for example, 3~5mM, for example, 1mM, 3mM, 5mM or 7mM, example
Such as it is 3mM or 5mM, for example, 3mM.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710433656.6A CN107142239B (en) | 2017-06-09 | 2017-06-09 | Method for improving culture efficiency of bovine in vitro fertilization embryos |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710433656.6A CN107142239B (en) | 2017-06-09 | 2017-06-09 | Method for improving culture efficiency of bovine in vitro fertilization embryos |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107142239A true CN107142239A (en) | 2017-09-08 |
| CN107142239B CN107142239B (en) | 2020-01-17 |
Family
ID=59781099
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710433656.6A Active CN107142239B (en) | 2017-06-09 | 2017-06-09 | Method for improving culture efficiency of bovine in vitro fertilization embryos |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107142239B (en) |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107523538A (en) * | 2017-09-30 | 2017-12-29 | 江苏农林职业技术学院 | A kind of placenta in vitro culture method |
| CN108728404A (en) * | 2018-06-07 | 2018-11-02 | 天津博裕力牧科技有限公司 | Ox live body or the method for in vitro oocyte collection IVF Embryos culture |
| CN108728401A (en) * | 2018-06-07 | 2018-11-02 | 天津博裕力牧科技有限公司 | The method of IVF of Oocyte in Bovine Embryo Culture and culture medium used |
| CN108753689A (en) * | 2018-06-07 | 2018-11-06 | 天津博裕力牧科技有限公司 | The method of ox IVF Embryos culture |
| CN109321451A (en) * | 2018-10-11 | 2019-02-12 | 常州市环境监测中心 | It is a kind of can multiple groups experiment zebrafish embryo acute toxicity detection kit |
| CN109628382A (en) * | 2019-01-18 | 2019-04-16 | 西北农林科技大学 | A kind of culture solution and cultural method of the development quality improving ox embryo in vitro fertilization |
| CN114507636A (en) * | 2018-12-06 | 2022-05-17 | 中国农业大学 | Method for improving animal embryo development efficiency |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103898046A (en) * | 2014-02-28 | 2014-07-02 | 中国农业科学院北京畜牧兽医研究所 | Cow in-vitro fertilization embryo culture fluid and culture method thereof |
-
2017
- 2017-06-09 CN CN201710433656.6A patent/CN107142239B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103898046A (en) * | 2014-02-28 | 2014-07-02 | 中国农业科学院北京畜牧兽医研究所 | Cow in-vitro fertilization embryo culture fluid and culture method thereof |
Non-Patent Citations (2)
| Title |
|---|
| DO VH ET AL.: "Improvements to In Vitro Culture Media for Use in Bovine IVF", 《JOURNAL OF VETERINARY SCIENCE & ANIMAL HUSBANDRY》 * |
| LEVENT KESKINTEPE ET AL.: "Production of Viable Bovine Blastocysts in Defined In Vitro Conditions", 《BIOLOGY OF REPRODUCTION》 * |
Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107523538A (en) * | 2017-09-30 | 2017-12-29 | 江苏农林职业技术学院 | A kind of placenta in vitro culture method |
| CN107523538B (en) * | 2017-09-30 | 2020-08-25 | 江苏农林职业技术学院 | In-vitro culture method for embryo body |
| CN108728404A (en) * | 2018-06-07 | 2018-11-02 | 天津博裕力牧科技有限公司 | Ox live body or the method for in vitro oocyte collection IVF Embryos culture |
| CN108728401A (en) * | 2018-06-07 | 2018-11-02 | 天津博裕力牧科技有限公司 | The method of IVF of Oocyte in Bovine Embryo Culture and culture medium used |
| CN108753689A (en) * | 2018-06-07 | 2018-11-06 | 天津博裕力牧科技有限公司 | The method of ox IVF Embryos culture |
| CN108753689B (en) * | 2018-06-07 | 2021-09-07 | 天津博裕力牧科技有限公司 | Method for culturing bovine in-vitro fertilized embryo |
| CN108728401B (en) * | 2018-06-07 | 2021-09-10 | 天津博裕力牧科技有限公司 | Method for culturing bovine oocyte in vitro fertilization embryo and used culture medium |
| CN108728404B (en) * | 2018-06-07 | 2021-09-10 | 天津博裕力牧科技有限公司 | Method for culturing in vitro fertilization embryo of bovine living body or in vitro collected oocyte |
| CN109321451A (en) * | 2018-10-11 | 2019-02-12 | 常州市环境监测中心 | It is a kind of can multiple groups experiment zebrafish embryo acute toxicity detection kit |
| CN109321451B (en) * | 2018-10-11 | 2021-08-10 | 常州市环境监测中心 | Zebra fish embryo acute toxicity detection kit for multiple groups of experiments |
| CN114507636A (en) * | 2018-12-06 | 2022-05-17 | 中国农业大学 | Method for improving animal embryo development efficiency |
| CN109628382A (en) * | 2019-01-18 | 2019-04-16 | 西北农林科技大学 | A kind of culture solution and cultural method of the development quality improving ox embryo in vitro fertilization |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107142239B (en) | 2020-01-17 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107142239A (en) | The method for improving ox IVF Embryos culture efficiency | |
| CN107034173A (en) | The method of ox IVF Embryos culture and the nutrient solution used | |
| CN103898046B (en) | Ox IVF Embryos nutrient solution and cultural method | |
| Zhang et al. | Advances on in vitro production and cryopreservation of porcine embryos | |
| CN110066763A (en) | Promote the method for ox embryo in vitro culture development of fertilized ova | |
| WO2016206086A1 (en) | Pig oocyte in vitro maturation culture solution and preparation method and culture method thereof | |
| CN102140435A (en) | Method for improving in-vitro production efficiency of buffalo embryos | |
| CN101945992A (en) | Protein-free gamete and embryo handling and culture media products | |
| JP2019502372A (en) | Methods, media and products for culturing embryos | |
| CN103710299A (en) | In-vitro culture solution for culturing swine parthenogenetic activated embryos and swine in-vitro fertilized embryos | |
| KR100666595B1 (en) | Compositions of sequential synthetic culture media for in vitro maturation and fertilization of human oocyte and culture methods using thereof | |
| CN110066764A (en) | Promote the method for ox embryo in vitro culture oocyte in vitro maturation | |
| CN112048467A (en) | Application of KSOM-AA culture solution in-vitro culture of NOD (non-specific oligonucleotide) background mouse embryos | |
| CN109943522A (en) | A kind of mammalian in vitro fertilization Embryo Culture method | |
| CN108753689A (en) | The method of ox IVF Embryos culture | |
| CN108728404A (en) | Ox live body or the method for in vitro oocyte collection IVF Embryos culture | |
| CN108728401A (en) | The method of IVF of Oocyte in Bovine Embryo Culture and culture medium used | |
| Chohan et al. | In vitro maturation, fertilization and early cleavage rates of bovine fetal oocytes | |
| CN109628382A (en) | A kind of culture solution and cultural method of the development quality improving ox embryo in vitro fertilization | |
| CN109897820B (en) | Method for delaying aging of in vitro cultured ovum | |
| CN101962626B (en) | Calf in vitro embryo culture solution | |
| CN107916249A (en) | Improve the nutrient solution and cultural method of the development quality of bovine somatic cells clone's embryo and embryo in vitro fertilization | |
| CN107043743B (en) | In-vitro maturation method of canine oocytes | |
| CN101555466B (en) | Sheep embryo in-vitro culture solution containing astragalus polysaccharide and culture method thereof | |
| CN108715829A (en) | A method of improving Human in vitro Fertilization embryo development rate |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| TA01 | Transfer of patent application right | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20170920 Address after: 300457 Tianjin City, Tanggu Development Zone, Haitong Street No. 3 Alexander center building A Intco Burson Marsteller Applicant after: SINICA Ltd. Applicant after: TIANJIN BOYU LIMU TECHNOLOGY CO.,LTD. Applicant after: TIANJIN ZHONGKE BOYA BIOLOGICAL BREEDING RESEARCH Co.,Ltd. Address before: 300457 Tianjin City, Tanggu Development Zone, Haitong Street No. 3 Alexander center building A Intco Burson Marsteller Applicant before: SINICA Ltd. |
|
| TA01 | Transfer of patent application right | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20180913 Address after: 300457 A building, Alexander center, 3 Haitong street, Tianjin Binhai New Area. Applicant after: TIANJIN BOYU LIMU TECHNOLOGY CO.,LTD. Applicant after: TIANJIN ZHONGKE BOYA BIOLOGICAL BREEDING RESEARCH Co.,Ltd. Applicant after: TIANJIN LIMUD DINGFENG BIOTECHNOLOGY Co.,Ltd. Address before: 300457 Tianjin city Tanggu District Development Zone Haitong Street 3 Alexander center A floor, British Boya company Applicant before: SINICA Ltd. Applicant before: TIANJIN BOYU LIMU TECHNOLOGY CO.,LTD. Applicant before: TIANJIN ZHONGKE BOYA BIOLOGICAL BREEDING RESEARCH Co.,Ltd. |
|
| TA01 | Transfer of patent application right | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20190917 Address after: 300457 A building, Alexander center, 3 Haitong street, Tianjin Binhai New Area. Applicant after: TIANJIN BOYU LIMU TECHNOLOGY CO.,LTD. Address before: 300457 A building, Alexander center, 3 Haitong street, Tianjin Binhai New Area. Applicant before: TIANJIN BOYU LIMU TECHNOLOGY CO.,LTD. Applicant before: TIANJIN ZHONGKE BOYA BIOLOGICAL BREEDING RESEARCH Co.,Ltd. Applicant before: TIANJIN LIMUD DINGFENG BIOTECHNOLOGY Co.,Ltd. |
|
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20200311 Address after: 214092, Boya stem cell company, 88 Meiliang Road, Mashan, Wuxi, Jiangsu Patentee after: Wuxi boyulimu Biotechnology Co.,Ltd. Address before: 300457 A building, Alexander center, 3 Haitong street, Tianjin Binhai New Area. Patentee before: TIANJIN BOYU LIMU TECHNOLOGY CO.,LTD. |
|
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20240104 Address after: 300457 A building, Alexander center, 3 Haitong street, Tianjin Binhai New Area. Patentee after: Tianjin Limu Biotechnology Co.,Ltd. Address before: Boya stem cell company, No.88, Mashan Meiliang Road, Binhu District, Wuxi City, Jiangsu Province, 214092 Patentee before: Wuxi boyulimu Biotechnology Co.,Ltd. |