CN117770234A - Donkey sperm low-temperature preservation diluent taking donkey milk as nutrient solution - Google Patents
Donkey sperm low-temperature preservation diluent taking donkey milk as nutrient solution Download PDFInfo
- Publication number
- CN117770234A CN117770234A CN202311845686.XA CN202311845686A CN117770234A CN 117770234 A CN117770234 A CN 117770234A CN 202311845686 A CN202311845686 A CN 202311845686A CN 117770234 A CN117770234 A CN 117770234A
- Authority
- CN
- China
- Prior art keywords
- donkey
- milk
- diluent
- sperm
- nutrient solution
- 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.)
- Pending
Links
- 241000283074 Equus asinus Species 0.000 title claims abstract description 119
- 239000003085 diluting agent Substances 0.000 title claims abstract description 80
- 235000020250 donkey milk Nutrition 0.000 title claims abstract description 70
- 238000004321 preservation Methods 0.000 title claims abstract description 62
- 235000015097 nutrients Nutrition 0.000 title claims abstract description 26
- 210000002969 egg yolk Anatomy 0.000 claims abstract description 36
- 238000002360 preparation method Methods 0.000 claims abstract description 34
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 claims abstract description 24
- 239000000725 suspension Substances 0.000 claims abstract description 18
- 238000007710 freezing Methods 0.000 claims abstract description 16
- 230000008014 freezing Effects 0.000 claims abstract description 15
- 238000000034 method Methods 0.000 claims abstract description 14
- 238000004806 packaging method and process Methods 0.000 claims abstract description 13
- 229960005322 streptomycin Drugs 0.000 claims abstract description 12
- 229930182555 Penicillin Natural products 0.000 claims abstract description 10
- 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 claims abstract description 10
- 229940049954 penicillin Drugs 0.000 claims abstract description 10
- 238000002156 mixing Methods 0.000 claims abstract description 6
- 102000002322 Egg Proteins Human genes 0.000 claims description 24
- 108010000912 Egg Proteins Proteins 0.000 claims description 24
- 235000013345 egg yolk Nutrition 0.000 claims description 16
- 238000005138 cryopreservation Methods 0.000 claims description 15
- 238000001914 filtration Methods 0.000 claims description 12
- 230000001954 sterilising effect Effects 0.000 claims description 10
- 238000001816 cooling Methods 0.000 claims description 9
- 239000006260 foam Substances 0.000 claims description 9
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 claims description 8
- 210000000969 egg white Anatomy 0.000 claims description 8
- 235000014103 egg white Nutrition 0.000 claims description 8
- 238000009835 boiling Methods 0.000 claims description 6
- 210000002445 nipple Anatomy 0.000 claims description 6
- 238000005303 weighing Methods 0.000 claims description 5
- 208000035473 Communicable disease Diseases 0.000 claims description 4
- 240000001624 Espostoa lanata Species 0.000 claims description 4
- 235000009161 Espostoa lanata Nutrition 0.000 claims description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 4
- 238000001514 detection method Methods 0.000 claims description 4
- 238000007598 dipping method Methods 0.000 claims description 4
- 201000010099 disease Diseases 0.000 claims description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 4
- 210000003278 egg shell Anatomy 0.000 claims description 4
- 235000013601 eggs Nutrition 0.000 claims description 4
- 230000036541 health Effects 0.000 claims description 4
- 208000015181 infectious disease Diseases 0.000 claims description 4
- 210000005075 mammary gland Anatomy 0.000 claims description 4
- 238000005096 rolling process Methods 0.000 claims description 4
- 210000001534 vitelline membrane Anatomy 0.000 claims description 4
- 238000004140 cleaning Methods 0.000 claims description 3
- 238000005201 scrubbing Methods 0.000 claims description 3
- 229910001220 stainless steel Inorganic materials 0.000 claims description 3
- 239000010935 stainless steel Substances 0.000 claims description 3
- 239000000645 desinfectant Substances 0.000 claims description 2
- 230000006651 lactation Effects 0.000 claims description 2
- 239000007788 liquid Substances 0.000 claims description 2
- 235000016709 nutrition Nutrition 0.000 claims 1
- 230000009027 insemination Effects 0.000 abstract description 21
- 230000000694 effects Effects 0.000 abstract description 19
- 244000144972 livestock Species 0.000 abstract description 9
- 238000004519 manufacturing process Methods 0.000 abstract description 7
- 229940079593 drug Drugs 0.000 abstract description 6
- 239000003814 drug Substances 0.000 abstract description 6
- 239000003242 anti bacterial agent Substances 0.000 abstract description 5
- 229940088710 antibiotic agent Drugs 0.000 abstract description 5
- 238000009395 breeding Methods 0.000 abstract description 2
- 230000001488 breeding effect Effects 0.000 abstract description 2
- 239000000243 solution Substances 0.000 description 20
- 210000000582 semen Anatomy 0.000 description 18
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 15
- 230000019100 sperm motility Effects 0.000 description 10
- 241000283073 Equus caballus Species 0.000 description 7
- 238000005457 optimization Methods 0.000 description 7
- 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 description 6
- 239000008103 glucose Substances 0.000 description 6
- 230000033001 locomotion Effects 0.000 description 6
- 241001465754 Metazoa Species 0.000 description 5
- 238000010790 dilution Methods 0.000 description 5
- 239000012895 dilution Substances 0.000 description 5
- 229940088597 hormone Drugs 0.000 description 5
- 239000005556 hormone Substances 0.000 description 5
- 230000009467 reduction Effects 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- GVJHHUAWPYXKBD-UHFFFAOYSA-N (±)-α-Tocopherol Chemical compound OC1=C(C)C(C)=C2OC(CCCC(C)CCCC(C)CCCC(C)C)(C)CCC2=C1C GVJHHUAWPYXKBD-UHFFFAOYSA-N 0.000 description 4
- 230000012173 estrus Effects 0.000 description 4
- XOAAWQZATWQOTB-UHFFFAOYSA-N taurine Chemical compound NCCS(O)(=O)=O XOAAWQZATWQOTB-UHFFFAOYSA-N 0.000 description 4
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 239000007983 Tris buffer Substances 0.000 description 3
- 210000003022 colostrum Anatomy 0.000 description 3
- 235000021277 colostrum Nutrition 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 3
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 2
- 239000004475 Arginine Substances 0.000 description 2
- 229930091371 Fructose Natural products 0.000 description 2
- 239000005715 Fructose Substances 0.000 description 2
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 2
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 2
- MUPFEKGTMRGPLJ-RMMQSMQOSA-N Raffinose Natural products O(C[C@H]1[C@@H](O)[C@H](O)[C@@H](O)[C@@H](O[C@@]2(CO)[C@H](O)[C@@H](O)[C@@H](CO)O2)O1)[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 MUPFEKGTMRGPLJ-RMMQSMQOSA-N 0.000 description 2
- QNVSXXGDAPORNA-UHFFFAOYSA-N Resveratrol Natural products OC1=CC=CC(C=CC=2C=C(O)C(O)=CC=2)=C1 QNVSXXGDAPORNA-UHFFFAOYSA-N 0.000 description 2
- LUKBXSAWLPMMSZ-OWOJBTEDSA-N Trans-resveratrol Chemical compound C1=CC(O)=CC=C1\C=C\C1=CC(O)=CC(O)=C1 LUKBXSAWLPMMSZ-OWOJBTEDSA-N 0.000 description 2
- MUPFEKGTMRGPLJ-UHFFFAOYSA-N UNPD196149 Natural products OC1C(O)C(CO)OC1(CO)OC1C(O)C(O)C(O)C(COC2C(C(O)C(O)C(CO)O2)O)O1 MUPFEKGTMRGPLJ-UHFFFAOYSA-N 0.000 description 2
- 229930003427 Vitamin E Natural products 0.000 description 2
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 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 description 2
- 210000000170 cell membrane Anatomy 0.000 description 2
- 229960004407 chorionic gonadotrophin Drugs 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 239000012154 double-distilled water Substances 0.000 description 2
- WIGCFUFOHFEKBI-UHFFFAOYSA-N gamma-tocopherol Natural products CC(C)CCCC(C)CCCC(C)CCCC1CCC2C(C)C(O)C(C)C(C)C2O1 WIGCFUFOHFEKBI-UHFFFAOYSA-N 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 239000008101 lactose Substances 0.000 description 2
- 230000004899 motility Effects 0.000 description 2
- 210000001672 ovary Anatomy 0.000 description 2
- 239000001508 potassium citrate Substances 0.000 description 2
- 229960002635 potassium citrate Drugs 0.000 description 2
- QEEAPRPFLLJWCF-UHFFFAOYSA-K potassium citrate (anhydrous) Chemical compound [K+].[K+].[K+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O QEEAPRPFLLJWCF-UHFFFAOYSA-K 0.000 description 2
- 235000011082 potassium citrates Nutrition 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- LXNHXLLTXMVWPM-UHFFFAOYSA-N pyridoxine Chemical compound CC1=NC=C(CO)C(CO)=C1O LXNHXLLTXMVWPM-UHFFFAOYSA-N 0.000 description 2
- MUPFEKGTMRGPLJ-ZQSKZDJDSA-N raffinose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO[C@@H]2[C@@H]([C@@H](O)[C@@H](O)[C@@H](CO)O2)O)O1 MUPFEKGTMRGPLJ-ZQSKZDJDSA-N 0.000 description 2
- 235000021283 resveratrol Nutrition 0.000 description 2
- 229940016667 resveratrol Drugs 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 235000020183 skimmed milk Nutrition 0.000 description 2
- 239000001509 sodium citrate Substances 0.000 description 2
- 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 description 2
- DAEPDZWVDSPTHF-UHFFFAOYSA-M sodium pyruvate Chemical compound [Na+].CC(=O)C([O-])=O DAEPDZWVDSPTHF-UHFFFAOYSA-M 0.000 description 2
- 229960003080 taurine Drugs 0.000 description 2
- 235000019165 vitamin E Nutrition 0.000 description 2
- 239000011709 vitamin E Substances 0.000 description 2
- 229940046009 vitamin E Drugs 0.000 description 2
- IIZPXYDJLKNOIY-JXPKJXOSSA-N 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine Chemical class CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCC\C=C/C\C=C/C\C=C/C\C=C/CCCCC IIZPXYDJLKNOIY-JXPKJXOSSA-N 0.000 description 1
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 1
- CWVRJTMFETXNAD-FWCWNIRPSA-N 3-O-Caffeoylquinic acid Natural products O[C@H]1[C@@H](O)C[C@@](O)(C(O)=O)C[C@H]1OC(=O)\C=C\C1=CC=C(O)C(O)=C1 CWVRJTMFETXNAD-FWCWNIRPSA-N 0.000 description 1
- PZIRUHCJZBGLDY-UHFFFAOYSA-N Caffeoylquinic acid Natural products CC(CCC(=O)C(C)C1C(=O)CC2C3CC(O)C4CC(O)CCC4(C)C3CCC12C)C(=O)O PZIRUHCJZBGLDY-UHFFFAOYSA-N 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 235000005979 Citrus limon Nutrition 0.000 description 1
- 244000131522 Citrus pyriformis Species 0.000 description 1
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 1
- 101100136092 Drosophila melanogaster peng gene Proteins 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 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
- 244000068988 Glycine max Species 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- 239000007995 HEPES buffer Substances 0.000 description 1
- SQUHHTBVTRBESD-UHFFFAOYSA-N Hexa-Ac-myo-Inositol Natural products CC(=O)OC1C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C1OC(C)=O SQUHHTBVTRBESD-UHFFFAOYSA-N 0.000 description 1
- BVHLGVCQOALMSV-JEDNCBNOSA-N L-lysine hydrochloride Chemical compound Cl.NCCCC[C@H](N)C(O)=O BVHLGVCQOALMSV-JEDNCBNOSA-N 0.000 description 1
- CWVRJTMFETXNAD-KLZCAUPSSA-N Neochlorogenin-saeure Natural products O[C@H]1C[C@@](O)(C[C@@H](OC(=O)C=Cc2ccc(O)c(O)c2)[C@@H]1O)C(=O)O CWVRJTMFETXNAD-KLZCAUPSSA-N 0.000 description 1
- BYPFEZZEUUWMEJ-UHFFFAOYSA-N Pentoxifylline Chemical compound O=C1N(CCCCC(=O)C)C(=O)N(C)C2=C1N(C)C=N2 BYPFEZZEUUWMEJ-UHFFFAOYSA-N 0.000 description 1
- CWEZAWNPTYBADX-UHFFFAOYSA-N Procyanidin Natural products OC1C(OC2C(O)C(Oc3c2c(O)cc(O)c3C4C(O)C(Oc5cc(O)cc(O)c45)c6ccc(O)c(O)c6)c7ccc(O)c(O)c7)c8c(O)cc(O)cc8OC1c9ccc(O)c(O)c9 CWEZAWNPTYBADX-UHFFFAOYSA-N 0.000 description 1
- 229930003779 Vitamin B12 Natural products 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 238000012271 agricultural production Methods 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 238000009411 base construction Methods 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 235000001368 chlorogenic acid Nutrition 0.000 description 1
- 229940074393 chlorogenic acid Drugs 0.000 description 1
- CWVRJTMFETXNAD-JUHZACGLSA-N chlorogenic acid Chemical compound O[C@@H]1[C@H](O)C[C@@](O)(C(O)=O)C[C@H]1OC(=O)\C=C\C1=CC=C(O)C(O)=C1 CWVRJTMFETXNAD-JUHZACGLSA-N 0.000 description 1
- FFQSDFBBSXGVKF-KHSQJDLVSA-N chlorogenic acid Natural products O[C@@H]1C[C@](O)(C[C@@H](CC(=O)C=Cc2ccc(O)c(O)c2)[C@@H]1O)C(=O)O FFQSDFBBSXGVKF-KHSQJDLVSA-N 0.000 description 1
- BMRSEYFENKXDIS-KLZCAUPSSA-N cis-3-O-p-coumaroylquinic acid Natural products O[C@H]1C[C@@](O)(C[C@@H](OC(=O)C=Cc2ccc(O)cc2)[C@@H]1O)C(=O)O BMRSEYFENKXDIS-KLZCAUPSSA-N 0.000 description 1
- 235000015165 citric acid Nutrition 0.000 description 1
- AGVAZMGAQJOSFJ-WZHZPDAFSA-M cobalt(2+);[(2r,3s,4r,5s)-5-(5,6-dimethylbenzimidazol-1-yl)-4-hydroxy-2-(hydroxymethyl)oxolan-3-yl] [(2r)-1-[3-[(1r,2r,3r,4z,7s,9z,12s,13s,14z,17s,18s,19r)-2,13,18-tris(2-amino-2-oxoethyl)-7,12,17-tris(3-amino-3-oxopropyl)-3,5,8,8,13,15,18,19-octamethyl-2 Chemical compound [Co+2].N#[C-].[N-]([C@@H]1[C@H](CC(N)=O)[C@@]2(C)CCC(=O)NC[C@@H](C)OP(O)(=O)O[C@H]3[C@H]([C@H](O[C@@H]3CO)N3C4=CC(C)=C(C)C=C4N=C3)O)\C2=C(C)/C([C@H](C\2(C)C)CCC(N)=O)=N/C/2=C\C([C@H]([C@@]/2(CC(N)=O)C)CCC(N)=O)=N\C\2=C(C)/C2=N[C@]1(C)[C@@](C)(CC(N)=O)[C@@H]2CCC(N)=O AGVAZMGAQJOSFJ-WZHZPDAFSA-M 0.000 description 1
- 239000006059 cover glass Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000001627 detrimental effect Effects 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 238000005485 electric heating Methods 0.000 description 1
- YEIKGFFXYDKWAJ-UHFFFAOYSA-N ethyl hydrogen sulfate;2-piperazin-1-ylethanol Chemical compound CCOS(O)(=O)=O.OCCN1CCNCC1 YEIKGFFXYDKWAJ-UHFFFAOYSA-N 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 229940087559 grape seed Drugs 0.000 description 1
- 229910021389 graphene Inorganic materials 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- CDAISMWEOUEBRE-GPIVLXJGSA-N inositol Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](O)[C@@H]1O CDAISMWEOUEBRE-GPIVLXJGSA-N 0.000 description 1
- 229960000367 inositol Drugs 0.000 description 1
- GOMNOOKGLZYEJT-UHFFFAOYSA-N isoflavone Chemical compound C=1OC2=CC=CC=C2C(=O)C=1C1=CC=CC=C1 GOMNOOKGLZYEJT-UHFFFAOYSA-N 0.000 description 1
- CJWQYWQDLBZGPD-UHFFFAOYSA-N isoflavone Natural products C1=C(OC)C(OC)=CC(OC)=C1C1=COC2=C(C=CC(C)(C)O3)C3=C(OC)C=C2C1=O CJWQYWQDLBZGPD-UHFFFAOYSA-N 0.000 description 1
- 235000008696 isoflavones Nutrition 0.000 description 1
- 230000013011 mating Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 210000001700 mitochondrial membrane Anatomy 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000016087 ovulation Effects 0.000 description 1
- 229960001476 pentoxifylline Drugs 0.000 description 1
- 229920002414 procyanidin Polymers 0.000 description 1
- 239000008213 purified water Substances 0.000 description 1
- RADKZDMFGJYCBB-UHFFFAOYSA-N pyridoxal hydrochloride Natural products CC1=NC=C(CO)C(C=O)=C1O RADKZDMFGJYCBB-UHFFFAOYSA-N 0.000 description 1
- CDAISMWEOUEBRE-UHFFFAOYSA-N scyllo-inosotol Natural products OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 description 1
- 235000002639 sodium chloride Nutrition 0.000 description 1
- 235000011083 sodium citrates Nutrition 0.000 description 1
- 229940054269 sodium pyruvate Drugs 0.000 description 1
- 239000000600 sorbitol Substances 0.000 description 1
- 238000003879 sperm preservation Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- DVBIMNUZCMCPRL-UHFFFAOYSA-K trisodium;2-hydroxypropane-1,2,3-tricarboxylate;pentahydrate Chemical compound O.O.O.O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O DVBIMNUZCMCPRL-UHFFFAOYSA-K 0.000 description 1
- 210000004291 uterus Anatomy 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 239000000273 veterinary drug Substances 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
- 235000019163 vitamin B12 Nutrition 0.000 description 1
- 239000011715 vitamin B12 Substances 0.000 description 1
- 235000019158 vitamin B6 Nutrition 0.000 description 1
- 239000011726 vitamin B6 Substances 0.000 description 1
- 229940011671 vitamin b6 Drugs 0.000 description 1
Landscapes
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention relates to the technical field of livestock breeding, in particular to donkey sperm low-temperature preservation diluent taking donkey milk as nutrient solution; the method comprises the following steps of: firstly, measuring and mixing the treated room-temperature donkey milk and yolk, adding penicillin and streptomycin, and shaking to obtain a suspension; step two, centrifuging the suspension to obtain a centrifugate; and thirdly, packaging the centrifugate, and freezing and preserving to obtain donkey sperm low-temperature preservation diluent taking donkey milk as a nutrient solution. The donkey sperm low-temperature preservation diluent consists of donkey milk, yolk and antibiotics, and compared with the prior art, the donkey sperm low-temperature preservation diluent greatly reduces the production cost, has long preservation time and good effect, and has conception rate superior to or equivalent to that of the prior art; meanwhile, the required equipment is simple, the types of medicines required for preparation are few, the preparation procedure is simple, the preparation difficulty is low, no higher professional skill is required for operators, and the work can be independently completed in a common farm or a basic-level artificial insemination point; is suitable for popularization and application in large-scale farms and artificial insemination spots.
Description
Technical Field
The invention relates to the technical field of livestock breeding, in particular to donkey sperm low-temperature preservation diluent taking donkey milk as a nutrient solution.
Background
The research on the low-temperature and cryopreservation of equine sperm cells at home and abroad has been long-lived. At present, research reports and patent literature on equine animal sperm preservation diluents are available at home and abroad.
Foreign report: 20% of the donkey colostrum diluent reported by the agricultural institute of Spain Sarogosa Ala in 2019 and the center of the military for horse reproduction, cristina Á lvarez, victoria Lu ñ o, noelia Gonz lez and the like has good effect on donkey sperm freezing (Criolog, 2019, 87:110-114), 20% of the donkey colostrum diluent reported in 7 months in the same year has better protection effect on donkey sperm freezing than 20% of the horse colostrum and 20% of the yolk diluent, and various indexes such as total sperm viability, movement speed and the like are obviously improved after frozen and thawed sperm (Reproduction in Domestic Animals,2019, 7:94-97). The addition of egg yolk centrifugate to sperm freezing dilutions was reported by Argentina Ailisi university, 2018, alejandro Ferrante, clara Baca Castex, santiago Bruno et al to have a detrimental effect on sperm motility, membrane function and acrosome status (Journal of Equine Veterinary Science,2018, 65:12-18). Sorbitol was reported by the university of Spanish, coladow, veterinary drug institute J.Dorado, M.Hidalgo, D.Acha et al in 2019 as an alternative to improving sperm quality (Animal Reproduction Science,2019, 208:1-9).
Foreign patent products: foreign equine dilutions products have become popular such as the IRA series of IMV in france, gent A in Germany, E-Z Mixin dilutions in the U.S. INRA82 and INRA96 dilutions in France have been commercially sold by the French IMV Kasu company, and are formulated primarily with sugar salts and other additional ingredients.
Domestic reports: after semen centrifugation, the semen was stored at 5℃with INRA82 (manufactured by Kasu technologies Co., ltd., france) for an effective storage time of not more than 72 hours. The INRA82 dilution contains saccharides (glucose, lactose and raffinose), buffer substances (sodium citrate, potassium citrate and hydroxyethyl piperazine ethyl sulfate), anti-freezing agent (skimmed milk), antibiotics (penicillin and streptomycin) and other substances (Heilongjiang animal doctor, 2020, (38) 20:32-35). The addition of resveratrol and chlorogenic acid in semen diluent can significantly improve sperm quality and improve low-temperature preservation effect of donkey semen (Chinese journal of livestock, 2022, (58) 5:153-157). Adding 40 mu m resveratrol and 5 mu m MnCl into the refrigerating fluid 2 Can improve the frozen semen quality of donkey, remarkably improve the sperm motility, plasma membrane integrity and acrosome integrity after freezing and thawing, reduce the ROS content and improve the antioxidant enzyme activity (Jilin livestock veterinarian, 2021, 12:1-3). The addition amounts of the citric acid, the glucose and the yolk are respectively 17.0 g/L, 15.0g/L and 15 percent, and the donkey semen preservation effect at normal temperature is better (Heilongjiang livestock veterinarian, 2018, 13:102-104). The Tris-based diluent has better preservation and freezing effects on donkey sperm at normal temperature (Heilongjiang animal doctor, 2017, (09 below): 83-86). The filtration mode of adding yolk after the filtration of the base solution, the addition of 200 mu g/100 ml or 300 mu g/100 ml VC diluent, the addition of 5% VE diluent and the addition of 30% graphene oxide diluent have good low-temperature preservation effect on donkey sperms (Tianjin academy of agriculture, 2020, (27) 1:53-57). Zhang Wenqian, wang Lu, zhang Jiang et al report that the addition of 0.06g/L grape seed procyanidins to the diluent helps to improve the cryopreservation quality of donkey semen (Heilongjiang livestock veterinarian, 2022, (140) 17:52-55). Han Yingxue, gao Peng, li Nan and the like report that the addition of vitamin E with concentration of 1%, 2%, 4% and 6% in the diluent can improve the kinetic parameters, mitochondrial membrane potential, acrosome integrity and DNA integrity after donkey sperm are frozen and thawed and reduce the deformity rate, wherein 2% of vitamin E has the best effect (Chinese journal of livestock, 2022, (58) 9:179-183). Zhao Juan, li Jiandong report that arginine and BSA have a certain degree of improvement on sperm motility, acrosome integrity and plasma membrane integrity of donkey semen when added togetherHas a certain synergistic effect, and the optimal combination concentration is 1.0mg/mL of arginine and 2mg/mL of BSA (Heilongjiang livestock veterinarian, 2022, (141) 18:68-72).
Domestic patent literature: 1) The donkey sperm diluent, the preparation method and the application thereof (CN 111493062A) applied by Taidong (Zhenjiang) biotechnology Co-Ltd in 2020, comprises the following components in mass: 30.4+/-0.5 g of Tris, 17.0+/-0.2 g of citric acid, 7.5+/-0.1 g of glucose, 12.5+/-0.2 mg of taurine, 36.3+/-0.5 mg of sodium pyruvate, 1.5+/-0.1 g of fructose, 2+/-0.1 g of modified lecithin, 1.5+/-0.1 g of pentoxifylline and 25.4+/-0.3 mg of soybean isoflavone. 2) A donkey frozen semen diluent formula and a preparation method thereof (CN 111374124A) applied by a modern agricultural production base construction engineering center of Liaoning province in 2020 comprises 1.2g of glucose, 1.3g of citric acid, 2.4g of Tris2.4g of glycerol, 5ml of double distilled water, 100ml of yolk, 20ml of penicillin 1X 105IU and 1X 105IU of streptomycin. 3) In 2019, shandong livestock veterinary professional college applied for a donkey semen diluent and a preparation method thereof (CN 110583630A), the donkey semen diluent comprises glucose, lactose, raffinose, sodium citrate pentahydrate, potassium citrate, HEPES, EDTA, egg yolk, green streptomycin, sterile distilled water, defatted donkey milk, and BSA and A solution, wherein the A solution comprises taurine, L-lysine hydrochloride, inositol, vitamin B6, vitamin B12, citric acid, sodium citrate, lemon yellow and purified water. 4) In 2017 Tianjin agricultural institute application of a method for preserving donkey semen at low temperature (CN 107183013A), a base solution is prepared according to the mass parts of 3.04 parts of Tris, 1.70 parts of citric acid, 1.50 parts of glucose, 1.50 parts of fructose, 0.06 part of penicillin, 0.10 part of streptomycin and 100 parts of double distilled water, and 85 parts of the base solution and 15 parts of egg yolk solution are used for preparing a semen diluent.
Besides the above foreign and domestic reports and patents, some of the prior reported and declared donkey sperm diluent patent literature and horse sperm diluent patent literature commonly adopt the above sugar salts, skimmed milk, egg yolk and bioactive substances and other additive components as main components for preparing the diluent.
However, the donkey sperm low-temperature preservation diluent in the prior art has short preservation time, more instruments and equipment are needed for preparation, higher expertise is needed for operators, the preparation procedure is complicated, the preparation difficulty is high, the cost is high, and the work cannot be independently completed at a farm or a basic artificial insemination point, so that the requirements cannot be met.
Disclosure of Invention
The invention provides a donkey sperm low-temperature preservation diluent taking donkey milk as a nutrient solution, which overcomes the defects of the prior art, and can effectively solve the problems that the donkey sperm low-temperature preservation diluent in the prior art has short preservation time, needs more instruments and equipment for preparation, has higher professional skills for operators, has complicated preparation procedures, large preparation difficulty and high cost, and cannot independently complete the work at a plant or a basic artificial insemination point, and cannot meet the requirements.
The technical scheme of the invention is realized by the following measures: a donkey sperm low-temperature preservation diluent taking donkey milk as a nutrient solution is obtained by the following steps: firstly, weighing 80ml to 400ml of treated room temperature donkey milk, mixing with 20ml to 100ml of yolk, adding 10 to 50 ten thousand IU of penicillin and 10 to 50 ten thousand IU of streptomycin, and repeatedly oscillating for 30 to 50 minutes in a container with a cover to obtain suspension; step two, centrifuging the suspension for 2 to 3 times, wherein each time lasts for 10 to 20 minutes, and sucking the centrifugate after each treatment and repeating the operation to obtain centrifugate; and thirdly, packaging the centrifugate, and freezing and preserving to obtain donkey sperm low-temperature preservation diluent taking donkey milk as a nutrient solution.
The following are further optimizations and/or improvements to the above-described inventive solution:
in the preparation method of the donkey sperm low-temperature preservation diluent, in the second step, a 50ml centrifuge tube is used for subpackaging the suspension and centrifuging the suspension at a rotating speed of 2200 rpm; in the third step, packaging according to the dosage of 100 ml/bottle, and freezing and preserving after packaging.
The treated room temperature donkey milk is obtained by the following steps: firstly, scrubbing and cleaning the nipple of the donkey, sterilizing, milking, and filtering the extruded fresh donkey milk; secondly, placing the filtered fresh donkey milk into a sterilized container, boiling, skimming the swelled foam, and continuously 2 to 4 times until no foam swells, and covering a cover for cooling; and thirdly, cooling to room temperature, and filtering to obtain the treated room temperature donkey milk.
In the preparation method of the treated room temperature donkey milk, in the first step, the nipple of the donkey with lactation is scrubbed cleanly by a towel, and is milked after being disinfected by a special disinfectant, and the extruded fresh donkey milk is filtered by 6 layers of sterilizing gauze; in the second step, the filtered fresh donkey milk is placed in a sterilized stainless steel container, and the swelled foam is skimmed by boiling in an electromagnetic oven; and in the third step, cooling to room temperature, and filtering with 6 layers of sterilized gauze to obtain the treated room temperature donkey milk.
The yolk is obtained by the following steps: selecting 2 to 10 fresh eggs from 3 days to 5 days, wiping and sterilizing the egg shells with alcohol cotton balls, airing, gently knocking the shells with forceps, pouring egg white out, rolling the egg yolk on filter paper, dipping the egg white, and inserting 20ml to 100ml of egg yolk into a yolk membrane by using a syringe with a needle removed to obtain the egg yolk.
The donkey milk is secreted by the donkey after delivery for more than 15 days; the donkey is a donkey without infectious disease or mammary gland disease and is qualified by quarantine and health detection.
The preparation process of the donkey sperm low-temperature preservation diluent is carried out under aseptic conditions.
The donkey sperm low-temperature preservation diluent consists of donkey milk, yolk and antibiotics, and compared with the prior art, the donkey sperm low-temperature preservation diluent greatly reduces the production cost, has long preservation time and good effect, and has conception rate superior to or equivalent to that of the prior art; meanwhile, compared with the existing donkey sperm low-temperature preservation diluent, the preparation method has the advantages of simple equipment, few types of medicines required for preparation, simple preparation procedure, low preparation difficulty, no need of higher professional skills of operators, and independent completion of the work in general farms or basic-level artificial insemination points; is suitable for popularization and application in large-scale farms and artificial insemination spots.
Description of the embodiments
The present invention is not limited by the following examples, and specific embodiments can be determined according to the technical scheme and practical situations of the present invention.
Example 1, the donkey sperm low-temperature preservation diluent with donkey milk as nutrient solution is obtained by the following steps: firstly, weighing 80ml to 400ml of treated room temperature donkey milk, mixing with 20ml to 100ml of yolk, adding 10 to 50 ten thousand IU of penicillin and 10 to 50 ten thousand IU of streptomycin, and repeatedly oscillating for 30 to 50 minutes in a container with a cover to obtain suspension; step two, centrifuging the suspension for 2 to 3 times, wherein each time lasts for 10 to 20 minutes, and sucking the centrifugate after each treatment and repeating the operation to obtain centrifugate; and thirdly, packaging the centrifugate, and freezing and preserving to obtain donkey sperm low-temperature preservation diluent taking donkey milk as a nutrient solution. The donkey sperm low-temperature preservation diluent consists of donkey milk, yolk and antibiotics, and compared with the prior art, the donkey sperm low-temperature preservation diluent greatly reduces the production cost, has long preservation time and good effect, and has conception rate superior to or equivalent to that of the prior art; meanwhile, compared with the existing donkey sperm low-temperature preservation diluent, the preparation method has the advantages of simple equipment, few types of medicines required for preparation, simple preparation procedure, low preparation difficulty, no need of higher professional skills of operators, and independent completion of the work in general farms or basic-level artificial insemination points; is suitable for popularization and application in large-scale farms and artificial insemination spots. The donkey sperm low-temperature preservation diluent has long low-temperature preservation time and good effect on donkey sperm, is preserved for 5 to 6 days in a refrigerator at the temperature of 2 to 7 ℃, has little reduction of sperm motility, and still keeps a faster movement speed; sperm motility decreased significantly from day 7 and the rate of motility slowed until day 14 with swinging sperm. The conception rate during estrus is about 60% on average due to factors such as the fatness of the donkey, the foal time, the insemination dosage and the like when the ovulation-promoting hormone is matched for use.
Example 2, the donkey sperm low-temperature preservation diluent with donkey milk as nutrient solution is obtained by the following steps: firstly, 80ml of treated room temperature donkey milk is measured and mixed with 20ml of yolk, 10 ten thousand IU of penicillin and 10 ten thousand IU of streptomycin are added, and the mixture is repeatedly vibrated for 30 minutes in a container with a cover to obtain suspension; step two, centrifuging the suspension for 2 times, each for 10 minutes, and sucking the centrifugate after each treatment to repeatedly operate to obtain centrifugate; and thirdly, packaging the centrifugate, and freezing and preserving to obtain donkey sperm low-temperature preservation diluent taking donkey milk as a nutrient solution. The donkey sperm low-temperature preservation diluent in the embodiment 2 has long low-temperature preservation time and good effect on donkey sperm, is preserved for 6 days in a refrigerator at 7 ℃, has little reduction of sperm motility, and still keeps a faster movement speed; the conception rate of the female donkey can reach 65% due to factors such as the fatness of the female donkey, the time of foal production, the insemination dosage and the like when the female donkey is matched with the ovulation-promoting hormone.
Example 3, the donkey sperm low-temperature preservation diluent with donkey milk as nutrient solution is obtained by the following steps: firstly, weighing 400ml of treated room temperature donkey milk and 100ml of yolk, mixing, adding 50 ten thousand IU of penicillin and 50 ten thousand IU of streptomycin, and repeatedly oscillating for 35 minutes in a container with a cover to obtain suspension; step two, centrifuging the suspension for 3 times, 15 minutes each time, and sucking the centrifugate after each treatment to repeatedly operate to obtain centrifugate; and thirdly, packaging the centrifugate, and freezing and preserving to obtain donkey sperm low-temperature preservation diluent taking donkey milk as a nutrient solution. The donkey sperm low-temperature preservation diluent in the embodiment 3 has long low-temperature preservation time and good effect on donkey sperm, and the donkey sperm has little reduction of sperm motility after being preserved in a refrigerator at 5 ℃ for 5 days, and still keeps a faster movement speed; the conception rate of the female donkey can reach 68% due to factors such as the fatness of the female donkey, the time of foal production, the insemination dosage and the like when the female donkey is matched with the ovulation-promoting hormone.
Example 4 the donkey sperm low-temperature preservation diluent with donkey milk as nutrient solution is obtained by the following steps: firstly, weighing 240ml of treated room temperature donkey milk and 60ml of yolk, mixing, adding 30 ten thousand IU of penicillin and 30 ten thousand IU of streptomycin, and repeatedly oscillating for 40 minutes in a container with a cover to obtain suspension; step two, centrifuging the suspension for 3 times, 15 minutes each time, and sucking the centrifugate after each treatment to repeatedly operate to obtain centrifugate; and thirdly, packaging the centrifugate, and freezing and preserving to obtain donkey sperm low-temperature preservation diluent taking donkey milk as a nutrient solution. The donkey sperm low-temperature preservation diluent in the embodiment 4 has long low-temperature preservation time and good effect on donkey sperm, is preserved for 5 days in a refrigerator at the temperature of 2 ℃, has little reduction of sperm motility, and still keeps a faster movement speed; the conception rate of the female donkey can reach 69% due to factors such as the fat condition of the female donkey, the foal time, the insemination dosage and the like when the female donkey is matched with the ovulation-promoting hormone.
Example 5 as an optimization of the above example, in the preparation method of donkey sperm cryopreservation diluent, in the second step, the suspension was dispensed with 50ml centrifuge tubes and centrifuged at 2200 rpm; in the third step, packaging according to the dosage of 100 ml/bottle, and freezing and preserving after packaging.
Example 6 as an optimization of the above example, the treated room temperature donkey milk was obtained as follows: firstly, scrubbing and cleaning the nipple of the donkey, sterilizing, milking, and filtering the extruded fresh donkey milk; secondly, placing the filtered fresh donkey milk into a sterilized container, boiling, skimming the swelled foam, and continuously 2 to 4 times until no foam swells, and covering a cover for cooling; and thirdly, cooling to room temperature, and filtering to obtain the treated room temperature donkey milk.
Example 7, as an optimization of the above example, in the preparation method of the treated room temperature donkey milk, in the first step, the nipple of the lactating donkey is scrubbed clean with a towel, sterilized with a special sterilizing liquid and then milked, and the extruded fresh donkey milk is filtered with 6 layers of sterilizing gauze; in the second step, the filtered fresh donkey milk is placed in a sterilized stainless steel container, and the swelled foam is skimmed by boiling in an electromagnetic oven; and in the third step, cooling to room temperature, and filtering with 6 layers of sterilized gauze to obtain the treated room temperature donkey milk.
Example 8, as an optimization of the above example, the yolk was obtained as follows: selecting 2 to 10 fresh eggs from 3 days to 5 days, wiping and sterilizing the egg shells with alcohol cotton balls, airing, gently knocking the shells with forceps, pouring egg white out, rolling the egg yolk on filter paper, dipping the egg white, and inserting 20ml to 100ml of egg yolk into a yolk membrane by using a syringe with a needle removed to obtain the egg yolk.
Example 9, as an optimization of the above example, donkey milk was secreted by the donkey for 15 or more days post partum; the donkey is a donkey without infectious disease or mammary gland disease and is qualified by quarantine and health detection.
Example 10 as an optimization of the above example, donkey milk is fresh donkey milk.
The invention relates to a method for using donkey sperm low-temperature preservation diluent
40min before semen collection by a pseudo-vagina method, adjusting the water temperature of a water bath kettle to 37 ℃, and putting the donkey sperm low-temperature preservation diluent into low-temperature preservation. The microscope is started to carry a heating plate, and the cover glass, the glass slide and the beaker to be filled with semen are preheated.
After semen collection and filtration, a 50ml centrifuge tube is used for split charging and centrifuging for 10 minutes at the rotating speed of 2200 rpm, the supernatant is discarded, the donkey sperm of the invention with the volume equal to that of the original semen is added into the low-temperature preservation diluent, and after the donkey sperm is subjected to microscopic examination, the donkey sperm is wrapped in a plurality of layers by towels and is preserved in a refrigerator at the temperature of 2 ℃. During artificial insemination, the inhalation injector is connected with the insemination jacket for donkey to perform artificial insemination on oestrus female donkey, and 5ml to 10ml of diluted semen is input into each donkey uterus.
Drug and device sources
The donkey milk and yolk used for storing the diluent at low temperature are self-made, and the green streptomycin is produced by Sichuan Dingjian pharmaceutical industry Limited company; human chorionic gonadotrophin (hCG) is produced by Ningbo Sansheng biotechnology Co.
An electric heating constant temperature water bath (model DZKW-D-2) produced by Yongguangming medical instrument Co., ltd; a bench type low-speed centrifuge (model TDZ 4-WS), manufactured by Hunan Instrument laboratory instruments development Co., ltd; the B-type ultrasonic diagnostic apparatus for animals is produced by Zhengzhou Boxiang electronic technology Co., ltd; the model of the biological microscope is XSP-T36B-1600X.
Technical effect verification of the invention
The donkey sperm low-temperature preservation diluent has long low-temperature preservation time and good effect on donkey sperm, is preserved for 5 to 6 days in a refrigerator at the temperature of 2 to 7 ℃, has little reduction of sperm motility, and still keeps a faster movement speed; sperm motility decreased significantly from day 7 and the rate of motility slowed until day 14 with swinging sperm. The conception rate during estrus is about 60% on average due to factors such as the fatness of the donkey, the foal time, the insemination dosage and the like when the ovulation-promoting hormone is matched for use. The specific mating method comprises the following steps: the ovaries on both sides of the oestrus donkey are scanned through rectal examination and B ultrasonic scanning, the follicles develop to more than 3.0cm, the human chorionic gonadotrophin is injected into 5000IU for the first time after 12 hours, the seed is bred for the second time after 36 hours, and then the ovaries can be supplemented for 1 time at 48 hours or 60 hours according to the development condition of the follicles until ovulation.
From the production cost, the cost of the donkey sperm low-temperature preservation diluent per 100ml is about 10 yuan RMB according to the market price, and the market price of the donkey sperm low-temperature preservation diluent with the same volume is more than 100 yuan, so that the cost of the diluent can be saved by more than 10 times for donkey farms and artificial insemination points.
Compared with the existing donkey sperm low-temperature preservation diluent, the donkey sperm low-temperature preservation diluent provided by the invention has the advantages of simple equipment, less types of medicines required for preparation, simple preparation procedure, low preparation difficulty, no need of higher professional skills of operators, and capability of independently completing the work in a general farm or a basic-layer artificial insemination spot; the cost per unit volume is far lower than the selling price of donkey sperm diluent in the existing market; the most important is that the sperm obtains higher conception rate after low-temperature preservation and hybridization, and the conception rate is superior to or equivalent to the prior art; is suitable for popularization and application in large-scale farms and artificial insemination spots.
In conclusion, the donkey sperm low-temperature preservation diluent consists of donkey milk, yolk and antibiotics, and compared with the prior art, the donkey sperm low-temperature preservation diluent greatly reduces the production cost, has long preservation time and good effect, and has conception rate superior to or equivalent to that of the prior art; meanwhile, compared with the existing donkey sperm low-temperature preservation diluent, the preparation method has the advantages of simple equipment, few types of medicines required for preparation, simple preparation procedure, low preparation difficulty, no need of higher professional skills of operators, and independent completion of the work in general farms or basic-level artificial insemination points; is suitable for popularization and application in large-scale farms and artificial insemination spots.
The technical characteristics form the embodiment of the invention, have stronger adaptability and implementation effect, and can increase or decrease unnecessary technical characteristics according to actual needs so as to meet the requirements of different situations.
Claims (10)
1. A donkey sperm low-temperature preservation diluent taking donkey milk as a nutrient solution is characterized by being prepared by the following steps: firstly, weighing 80ml to 400ml of treated room temperature donkey milk, mixing with 20ml to 100ml of yolk, adding 10 to 50 ten thousand IU of penicillin and 10 to 50 ten thousand IU of streptomycin, and repeatedly oscillating for 30 to 50 minutes in a container with a cover to obtain suspension; step two, centrifuging the suspension for 2 to 3 times, wherein each time lasts for 10 to 20 minutes, and sucking the centrifugate after each treatment and repeating the operation to obtain centrifugate; and thirdly, packaging the centrifugate, and freezing and preserving to obtain donkey sperm low-temperature preservation diluent taking donkey milk as a nutrient solution.
2. The donkey sperm cryopreservation diluent using donkey milk as a nutrient solution according to claim 1, wherein in the preparation method of the donkey sperm cryopreservation diluent, in the second step, a 50ml centrifuge tube is used for subpackaging the suspension and centrifuging at a rotation speed of 2200 rpm; in the third step, packaging according to the dosage of 100 ml/bottle, and freezing and preserving after packaging.
3. Donkey sperm cryopreservation diluent with donkey milk as nutritional liquid according to claim 1 or 2, characterized in that the treated room temperature donkey milk is obtained by the following steps: firstly, scrubbing and cleaning the nipple of the donkey, sterilizing, milking, and filtering the extruded fresh donkey milk; secondly, placing the filtered fresh donkey milk into a sterilized container, boiling, skimming the swelled foam, and continuously 2 to 4 times until no foam swells, and covering a cover for cooling; and thirdly, cooling to room temperature, and filtering to obtain the treated room temperature donkey milk.
4. A donkey sperm cryopreservation diluent using donkey milk as a nutrient solution according to claim 3, wherein in the preparation method of the donkey milk at room temperature after treatment, in the first step, the nipple of the donkey which is used for lactation is scrubbed cleanly by a towel, is disinfected by a special disinfectant, is milked, and the extruded fresh donkey milk is filtered by 6 layers of sterile gauze; in the second step, the filtered fresh donkey milk is placed in a sterilized stainless steel container, and the swelled foam is skimmed by boiling in an electromagnetic oven; and in the third step, cooling to room temperature, and filtering with 6 layers of sterilized gauze to obtain the treated room temperature donkey milk.
5. Donkey sperm cryopreservation diluent using donkey milk as a nutrient solution according to claim 1 or 2, characterized in that the yolk is obtained by the following method: selecting 2 to 10 fresh eggs from 3 days to 5 days, wiping and sterilizing the egg shells with alcohol cotton balls, airing, gently knocking the shells with forceps, pouring egg white out, rolling the egg yolk on filter paper, dipping the egg white, and inserting 20ml to 100ml of egg yolk into a yolk membrane by using a syringe with a needle removed to obtain the egg yolk.
6. The donkey sperm cryopreservation diluent using donkey milk as a nutrient solution according to claim 4, wherein the yolk is obtained by the following method: selecting 2 to 10 fresh eggs from 3 days to 5 days, wiping and sterilizing the egg shells with alcohol cotton balls, airing, gently knocking the shells with forceps, pouring egg white out, rolling the egg yolk on filter paper, dipping the egg white, and inserting 20ml to 100ml of egg yolk into a yolk membrane by using a syringe with a needle removed to obtain the egg yolk.
7. The donkey sperm cryopreservation diluent taking donkey milk as a nutrient solution according to claim 1 or 2, which is characterized in that donkey milk is secreted by the donkey for more than 15 days after delivery; the donkey is a donkey without infectious disease or mammary gland disease and is qualified by quarantine and health detection.
8. The donkey sperm cryopreservation diluent taking donkey milk as a nutrient solution according to claim 6, which is characterized in that donkey milk is secreted by the donkey for more than 15 days after delivery; the donkey is a donkey without infectious disease or mammary gland disease and is qualified by quarantine and health detection.
9. The donkey sperm cryopreservation diluent using donkey milk as a nutrient solution according to claim 1 or 2, wherein the donkey sperm cryopreservation diluent is prepared under aseptic conditions.
10. The donkey sperm cryopreservation diluent using donkey milk as a nutrient solution according to claim 8, wherein the donkey sperm cryopreservation diluent is prepared under aseptic conditions.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311845686.XA CN117770234A (en) | 2023-12-29 | 2023-12-29 | Donkey sperm low-temperature preservation diluent taking donkey milk as nutrient solution |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311845686.XA CN117770234A (en) | 2023-12-29 | 2023-12-29 | Donkey sperm low-temperature preservation diluent taking donkey milk as nutrient solution |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117770234A true CN117770234A (en) | 2024-03-29 |
Family
ID=90381288
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311845686.XA Pending CN117770234A (en) | 2023-12-29 | 2023-12-29 | Donkey sperm low-temperature preservation diluent taking donkey milk as nutrient solution |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117770234A (en) |
-
2023
- 2023-12-29 CN CN202311845686.XA patent/CN117770234A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Eldridge-Panuska et al. | Establishment of pregnancies after serial dilution or direct transfer by vitrified equine embryos | |
| Perez-Marin et al. | Clinical approach to the repeat breeder cow syndrome | |
| Biradar et al. | Study on efficacy of cosynch and ovsynch protocols on fertility in repeat breeder buffaloes | |
| CN107980765B (en) | Semen diluent for improving low-temperature preservation quality of donkey semen and preparation method and application thereof | |
| AU4435100A (en) | Compositions for freezing dog sperm, method of freezing the dog sperm utilizing the compositions and artificial insemination method employing the frozen dog sperm | |
| Udeh et al. | Effects of type of extender and storage conditions on the motility of goat spermatozoa | |
| CN1712519A (en) | Liquid for preserving pig semen | |
| CN117770234A (en) | Donkey sperm low-temperature preservation diluent taking donkey milk as nutrient solution | |
| CN110279658A (en) | A kind of oxytocin injection and preparation method thereof | |
| Olurode et al. | Effects of storage temperature and extension media on motility of caprine spermatozoa | |
| CN106818709A (en) | Cattle freezing seminal fluid dilution preparation method | |
| CN113616781B (en) | Cefolonine-containing preparation for treating dairy cow mastitis in dry period and preparation method thereof | |
| CN110251660A (en) | Improved ox method for collecting ovum of living body | |
| CN107334731B (en) | Cattle imidocarb dipropionate injection and preparation method and application thereof | |
| Sayid | Advances in bovine follicular aspiration technique | |
| CN107427555A (en) | Healthy reproduction and suppress the composition and method of hyperketonemia using IL 8 to increase the output of milk and improve in mammal | |
| Gunay et al. | Influence of bull seminal plasma on post-thaw ram semen parameters and fertility | |
| KR20190059015A (en) | Preparation method for frozen semen of cow | |
| CN117770235A (en) | Fresh donkey essence diluent taking donkey milk as nutrient solution | |
| CN110438068B (en) | Method for improving in-vitro fertilization embryo development rate of river buffalo | |
| Setiawati et al. | Effect of Estrus Synchronization with Prostaglandins (PGF2Α) and Gonadothropin Realising Hormone (GNRH) on the Hematological Profile of Pasundan Heifers during Pregnancy | |
| CN107982532B (en) | Duck hepatitis A virus antigen-antibody complex vaccine and preparation method thereof | |
| López‐Gatius et al. | Pregnancies and Live Offspring Following Transfer of One‐Step Vitrified Bovine Embryos | |
| CN109223716A (en) | A kind of porcine epidemic diarrhea resisting, swine fever, pseudo- mad dog and tetrad Yolk antibody soluble powder of transmissible gastroenteritis and preparation method thereof | |
| Dolník et al. | Hysterotomy of a dairy cow with long-time macerated foetus |
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 |