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Biotechnology: Status and Uses

Biotechnology: Status and Uses. Animal Science 434 John J. Parrish. Reproductive Biotechnology's. Artificial insemination In vitro embryo production In vivo embryo production Embryo transfer Gender selection Genetic engineering Cloning. In Vitro Production of Embryos.

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Biotechnology: Status and Uses

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  1. Biotechnology: Status and Uses Animal Science 434 John J. Parrish

  2. Reproductive Biotechnology's • Artificial insemination • In vitro embryo production • In vivo embryo production • Embryo transfer • Gender selection • Genetic engineering • Cloning

  3. In Vitro Production of Embryos • Oocyte isolation and maturation • Sperm preparation • Sperm capacitation • Fertilization • Embryo Development

  4. Oocyte Aspiration

  5. Searching Isolation

  6. Culture 24 Hours Incubation • Maturation Medium • Tyrode’s • FSH, LH, Estradiol • Fetal Calf Serum 39°C, 5% CO2 in Air

  7. Cumulus Expansion Unexpanded Expanded

  8. Sperm Preparation Semen 45% Percoll 90% Percoll

  9. Fertilization

  10. Embryo Development 2-Cell 4-Cell 8-Cell Morula

  11. Embryo Development Blastocyst

  12. In Vivo Embryo Production • Normal cycling female • horses • Superovulated female • Cattle • Sheep • Goats • Deer • Humans

  13. PGF2a eCG orFSH Multiple Ovulations Estrus Estrus Progesterone From C.L. First Follicular Wave 10-12 Stimulating Follicular Development Multiple Ovulations eCGorFSH Ovulation Estrus Estrus Progesterone From C.L. 17

  14. Stimulating Follicular Development • eCG (PMSG) • Single injection • FSH • 8 injections • AM/PM • Decreasing doses

  15. Day 0 eCG Day 2 AM/PM PGF2a Estrus Day 3.5 to 5 Stimulating Follicular Development AI at 12 and 24 hr after coming into estrus

  16. Day 0-3.5 FSH AM/PM (8 injections) Estrus Day 3.5 to 5 Day 2 AM/PM PGF2a Stimulating Follicular Development AI at 12 and 24 hr after coming into estrus

  17. Embryo Recovery • Early to mid blastocyst • Day 6 to 7 (estrus, breeding = day 0) • Flush uterus • Surgical • Nonsurgical

  18. Procedure for Non-Surgical Embryo Flush

  19. Non-Surgical Embryo Flush

  20. Stage of Embryo at Recovery Tight Morula (day 5 - 7) Early Blastocyst (day 7 - 8) Blastocyst (day 7 - 9)

  21. Embryo Transfer • Recipient must be synchronized with donor or 1 day behind • Surgical • Flank • Nonsurgical • Similar to AI but going through diestrus cervix

  22. 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%)

  23. ET - Scheme

  24. Embryo Transfer Uses • Introduction of new genetics • Import/Export • Twinning • Coupling with other biotechnologies

  25. Embryo Sexing • Hy Antigen • Associated with male cells • PCR and Detection of Y and X DNA

  26. Hy-Antigen

  27. Octopus Springs - Yellowstone National Park Home of Thermus aquaticus - Taq Polymerase

  28. PCR Approach to Embryo Sexing Y X F M F F F F F F

  29. Sperm Gender Selection Selection of X or Y sperm

  30. PERCENT DNA DIFFERENCES BETWEEN X and Y CHROMOSOME Human 2.9 Cattle 3.8 Chinchilla 7.5 Turkey 0 X Chromosome has more DNA!!!

  31. Flow Cytometer Separation of X and Y Sperm Sperm Stained With DNA Sensitive Fluorescent Dye 9 0° 0° L A S E R - + + - Y s o r t X s o r t

  32. Flow Cytometer for Sperm Separation

  33. Sperm Gender Selection • Flow Cytometry • Only method that works! • Very few sperm recovered • Currently not suitable for AI use • Will be expensive and has reduced fertility

  34. Fetal Sexing OR Ultrasound Evaluation • Day 55 - 65

  35. Cloning Split morula

  36. Cloning by Nuclear Transfer Cycles are limited Only 3 - 4 cycles

  37. First Clones by Nuclear Transfer at the UW

  38. Cloning Uses • Production of identicals • Agricultural uses • Transgenics • Stem cell production and research

  39. Nuclear Clonning to Produce Stem Cells Cells of InterestGrown in Culture Dish Mature Oocyte enucleated and cell of interest fused Embyro?? Develops Inner Cell Mass Cells isolated and grown in culture

  40. Stem Cells Grown in Culture and Allowed to Differentiate

  41. Genetic Manipulation

  42. Transgenic for Growth Hormone

  43. Gene Transfer Using Micro-Injection of Pronuclei Less than 1% efficiency

  44. Gene Transfer Using Viral Transfection Better success but left with potential for viral replication

  45. Problems with Gene Transfer • Problem • Need to control site of gene insertion • Need to control number of gene copies inserted • Solution • Do genetic manipulation on cells in culture • Select correct cells for nuclear transfer and cloning

  46. Potential Uses of Genetic Manipulation • Production of spare body parts • Disease resistance • Increased production traits

  47. Gender Selected Semen

  48. Dairy Production in the Tropics • The F1 - Holstein X Native Cow best in milk production • Any cross to get F2 is not as good as F1 • Potential in vitro embryo production • Import Holstein oocytes from US • Use native semen • Produce F1 cross embryo and implant in an F1 cow

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