Biotechnology: Status and UsesReproductive Biotechnology'sIn Vitro Production of EmbryosOocyte AspirationSearchingCultureCumulus ExpansionSperm PreparationFertilizationEmbryo DevelopmentSlide 11In Vivo Embryo ProductionStimulating Follicular DevelopmentSlide 14Slide 15Slide 16Embryo RecoveryProcedure for Non-Surgical Embryo FlushNon-Surgical Embryo FlushStage of Embryo at RecoveryEmbryo TransferSuccess (pregnancy rate)ET - SchemeEmbryo Transfer UsesEmbryo SexingHy-AntigenPowerPoint PresentationPCR Approach to Embryo SexingSperm Gender SelectionPERCENT DNA DIFFERENCES BETWEEN X and Y CHROMOSOMESlide 31Flow Cytometer for Sperm SeparationSlide 33Fetal SexingCloningCloning by Nuclear TransferFirst Clones by Nuclear Transfer at the UWCloning UsesNuclear Clonning to Produce Stem CellsStem Cells Grown in Culture and Allowed to DifferentiateGenetic ManipulationTransgenic for Growth HormoneGene Transfer Using Micro-Injection of PronucleiGene Transfer Using Viral TransfectionProblems with Gene TransferPotential Uses of Genetic ManipulationSlide 47Dairy Production in the TropicsSlide 49Biotechnology: Status and UsesBiotechnology: Status and UsesAnimal Science 434John J. ParrishAnimal Science 434John J. ParrishReproductive Biotechnology'sReproductive Biotechnology's•Artificial insemination•In vitro embryo production•In vivo embryo production•Embryo transfer•Gender selection•Genetic engineering•Cloning•Artificial insemination•In vitro embryo production•In vivo embryo production•Embryo transfer•Gender selection•Genetic engineering•CloningIn Vitro Production of EmbryosIn Vitro Production of Embryos•Oocyte isolation and maturation•Sperm preparation•Sperm capacitation•Fertilization•Embryo Development•Oocyte isolation and maturation•Sperm preparation•Sperm capacitation•Fertilization•Embryo DevelopmentOocyte AspirationOocyte AspirationSearchingSearchingIsolationIsolationCultureCultureMaturation Medium•Tyrode’s•FSH, LH, Estradiol•Fetal Calf SerumMaturation Medium•Tyrode’s•FSH, LH, Estradiol•Fetal Calf Serum24 Hours Incubation24 Hours Incubation39°C, 5% CO2 in Air39°C, 5% CO2 in AirCumulus ExpansionCumulus ExpansionUnexpandedUnexpandedExpandedExpandedSperm PreparationSperm Preparation45% Percoll90% PercollSemenFertilizationEmbryo DevelopmentEmbryo Development2-Cell2-Cell4-Cell4-Cell8-Cell8-CellMorulaMorulaEmbryo DevelopmentEmbryo DevelopmentBlastocystBlastocystIn Vivo Embryo ProductionIn Vivo Embryo Production•Normal cycling female–horses•Superovulated female–Cattle–Sheep–Goats–Deer–Humans•Normal cycling female–horses•Superovulated female–Cattle–Sheep–Goats–Deer–HumansStimulating Follicular DevelopmentStimulating Follicular DevelopmentOvulationOvulationEstrusProgesteroneFrom C.L.eCGorFSHeCGorFSHEstrusMultipleOvulationsMultipleOvulationsEstrusEstruseCG orFSHeCG orFSHProgesterone From C.L.Progesterone From C.L.First FollicularWaveFirst FollicularWave10-1210-12MultipleOvulationsMultipleOvulationsPGF2PGF21717Stimulating Follicular DevelopmentStimulating Follicular Development•eCG (PMSG)–Single injection•FSH–8 injections –AM/PM–Decreasing doses•eCG (PMSG)–Single injection•FSH–8 injections –AM/PM–Decreasing dosesStimulating Follicular DevelopmentStimulating Follicular DevelopmentDay 0eCGDay 0eCGDay 2AM/PMPGF2Day 2AM/PMPGF2EstrusDay 3.5 to 5EstrusDay 3.5 to 5AI at 12 and 24 hr aftercoming into estrusAI at 12 and 24 hr aftercoming into estrusStimulating Follicular DevelopmentStimulating Follicular DevelopmentDay 2AM/PMPGF2Day 2AM/PMPGF2EstrusDay 3.5 to 5EstrusDay 3.5 to 5AI at 12 and 24 hr aftercoming into estrusAI at 12 and 24 hr aftercoming into estrusDay 0-3.5FSH AM/PM (8 injections)Day 0-3.5FSH AM/PM (8 injections)Embryo RecoveryEmbryo Recovery•Early to mid blastocyst–Day 6 to 7 (estrus, breeding = day 0)•Flush uterus–Surgical–Nonsurgical•Early to mid blastocyst–Day 6 to 7 (estrus, breeding = day 0)•Flush uterus–Surgical–NonsurgicalProcedure for Non-Surgical Embryo FlushProcedure for Non-Surgical Embryo FlushNon-Surgical Embryo FlushNon-Surgical Embryo FlushStage of Embryo at Recovery Stage of Embryo at Recovery Tight Morula (day 5 - 7)Tight Morula (day 5 - 7)Early Blastocyst (day 7 - 8)Early Blastocyst (day 7 - 8)Blastocyst (day 7 - 9)Blastocyst (day 7 - 9)Embryo TransferEmbryo Transfer•Recipient must be synchronized with donor or 1 day behind•Surgical–Flank•Nonsurgical–Similar to AI but going through diestrus cervix•Recipient must be synchronized with donor or 1 day behind•Surgical–Flank•Nonsurgical–Similar to AI but going through diestrus cervixSuccess (pregnancy rate)Success (pregnancy rate)•In vivo embryos–Fresh (60%)–Frozen (50%)•In Vitro embryos–Fresh (40 - 50%, sometimes 60% if transfer 2 embryos)–Frozen (30 - 40%)•In vivo embryos–Fresh (60%)–Frozen (50%)•In Vitro embryos–Fresh (40 - 50%, sometimes 60% if transfer 2 embryos)–Frozen (30 - 40%)ET - SchemeET - SchemeEmbryo Transfer UsesEmbryo Transfer Uses•Introduction of new genetics•Import/Export•Twinning•Coupling with other biotechnologies•Introduction of new genetics•Import/Export•Twinning•Coupling with other biotechnologiesEmbryo SexingEmbryo Sexing•Hy Antigen–Associated with male cells•PCR and Detection of Y and X DNA•Hy Antigen–Associated with male cells•PCR and Detection of Y and X DNAHy-AntigenOctopus Springs - Yellowstone National ParkOctopus Springs - Yellowstone National ParkHome of Thermus aquaticus - Taq PolymeraseHome of Thermus aquaticus - Taq PolymerasePCR Approach to Embryo SexingXYF M F F F F F FSperm Gender SelectionSperm Gender SelectionSelection of X or Y spermSelection of X or Y spermPERCENT DNA DIFFERENCES BETWEEN X and Y CHROMOSOMEPERCENT DNA DIFFERENCES BETWEEN X and Y CHROMOSOMEHuman 2.9Cattle 3.8Chinchilla 7.5Turkey 0Human 2.9Cattle 3.8Chinchilla 7.5Turkey 0X Chromosome has more DNA!!!X Chromosome has more DNA!!!++--+-LLAASSEERR90°0°XX ssoorrttYY ssoorrttSpermStainedWith DNASensitiveFluorescentDyeSpermStainedWith DNASensitiveFluorescentDyeFlow Cytometer Separation of X and Y SpermFlow Cytometer Separation of X and Y SpermFlow Cytometer for Sperm SeparationFlow Cytometer for Sperm SeparationSperm Gender SelectionSperm Gender Selection•Flow Cytometry–Only method
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