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Biotechnology: Status and Uses Animal Science 434 John J. Parrish Animal Science 434 John J. Parrish
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Reproductive Biotechnology's Artificial insemination In vitro embryo production In vivo embryo production Embryo transfer Gender selection Stem Cells Genetic engineering Cloning Artificial insemination In vitro embryo production In vivo embryo production Embryo transfer Gender selection Stem Cells Genetic engineering Cloning
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In 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 Development
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In Vivo Embryo Production Normal cycling female –horses Superovulated female –Cattle –Sheep –Goats –Deer –Humans Normal cycling female –horses Superovulated female –Cattle –Sheep –Goats –Deer –Humans
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Stimulating Follicular Development Estrus eCG or FSH Progesterone From C.L. Progesterone From C.L. First Follicular Wave First Follicular Wave 10-12 Multiple Ovulations Multiple Ovulations PGF 2
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Non- Surgical Embryo Flush Day 6 or 7
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Stage of Embryo at Recovery Tight Morula (day 5 - 7) Early Blastocyst (day 7 - 8) Blastocyst (day 7 - 9)
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ET - Scheme
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Embryo 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 DNA
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Hy-Antigen
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Octopus Springs - Yellowstone National Park Home of Thermus aquaticus - Taq Polymerase
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PCR Approach to Embryo Sexing X Y F M F F F F F F
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Sperm Gender Selection Selection of X or Y sperm
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PERCENT DNA DIFFERENCES BETWEEN X and Y CHROMOSOME Human 2.9 Cattle 3.8 Chinchilla 7.5 Turkey 0 Human 2.9 Cattle 3.8 Chinchilla 7.5 Turkey 0 X Chromosome has more DNA!!!
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+ + - - + - L L A A S S E E R R 90° X X s s o o r r t t Y Y s s o o r r t t Sperm Stained With DNA Sensitive Fluorescent Dye Sperm Stained With DNA Sensitive Fluorescent Dye Flow Cytometer Separation of X and Y Sperm
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Sperm Gender Selection Flow Cytometry –Only method that works! –Very few sperm recovered –Reduced fertility –Expense will limit use Flow Cytometry –Only method that works! –Very few sperm recovered –Reduced fertility –Expense will limit use
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Fetal Sexing OR Ultrasound Evaluation Day 55 - 65
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Cloning Split morula
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Cloning by Nuclear Transfer Cycles are limited Only 3 - 4 cycles
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Nuclear Cloning to Produce Stem Cells Cells of Interest Grown in Culture Dish Cells of Interest Grown in Culture Dish Mature Oocyte enucleated and cell of interest fused Embyro?? Develops Embyro?? Develops Inner Cell Mass Cells isolated and grown in culture Differentiate Potential Tissue: Neural Adipose Connective Heart Potential Tissue: Neural Adipose Connective Heart
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Stem Cells Grown in Culture and Allowed to Differentiate
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Benefits of Stem Cell Research 1.Regenerative medicine and tissue engineering In vitro systems: drug discovery, toxicology, diagnostic assays and cell culture reagents Biomedical research: unravel mechanisms of disease and human development
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Genetic Manipulation
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Transgenic for Growth Hormone
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Gene Transfer Using Micro- Injection of Pronuclei Less than 1% efficiency
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Gene Transfer Using Viral Transfection Better success but left with potential for viral replication
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