Biotechnology: Status and Uses Animal Science 434 John J. Parrish 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 Artificial insemination In vitro embryo production In vivo embryo production Embryo transfer Gender selection Genetic engineering Cloning

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

Oocyte Aspiration

Searching Isolation

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

Cumulus Expansion Unexpanded Expanded

Sperm Preparation 45% Percoll 90% Percoll Semen

Fertilization

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

Embryo Development Blastocyst

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

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 Multiple Ovulations Multiple Ovulations PGF 2  17

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

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

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 FSH AM/PM (8 injections) Day FSH AM/PM (8 injections)

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

Procedure for Non-Surgical Embryo Flush

Non- Surgical Embryo Flush

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

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

Success (pregnancy rate) In vivo embryos –Fresh (60%) –Frozen (50%) In Vitro embryos –Fresh ( %, sometimes 60% if transfer 2 embryos) –Frozen ( %) In vivo embryos –Fresh (60%) –Frozen (50%) In Vitro embryos –Fresh ( %, sometimes 60% if transfer 2 embryos) –Frozen ( %)

ET - Scheme

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

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

Hy-Antigen

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

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

Sperm Gender Selection Selection of X or Y sperm

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!!!

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

Flow Cytometer for Sperm Separation

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

Fetal Sexing OR Ultrasound Evaluation Day

Cloning Split morula

Cloning by Nuclear Transfer Cycles are limited Only cycles

First Clones by Nuclear Transfer at the UW

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

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

Stem Cells Grown in Culture and Allowed to Differentiate

Genetic Manipulation

Transgenic for Growth Hormone

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

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

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

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

Gender Selected Semen

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