1. Biotechnology: Status and Uses Animal Science 434 John J. Parrish 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 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 Oocyte isolation and maturation Sperm preparation Sperm capacitation Fertilization Embryo Development

4. Oocyte Aspiration

5. Searching Isolation

6. Culture Maturation Medium Tyrode’s FSH, LH, Estradiol Fetal Calf Serum Maturation Medium Tyrode’s FSH, LH, Estradiol Fetal Calf Serum 24 Hours Incubation 39°C, 5% CO 2 in Air

7. Cumulus Expansion Unexpanded Expanded

8. Sperm Preparation 45% Percoll 90% Percoll Semen

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 Normal cycling female –horses Superovulated female –Cattle –Sheep –Goats –Deer –Humans

13. Stimulating Follicular Development Ovulation Estrus Progesterone From C.L. eCG or FSH Estrus Multiple Ovulations Multiple Ovulations 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  17

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

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

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

17. Embryo 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 –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 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%) 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 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 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 X Y 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 Human 2.9 Cattle 3.8 Chinchilla 7.5 Turkey 0 X Chromosome has more DNA!!!

31. + + - - + - 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

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 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 Production of identicals –Agricultural uses –Transgenics –Stem cell production and research

39. Nuclear Clonning to Produce Stem Cells Cells of Interest Grown 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 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 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 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