Biotechnology: Status and Uses

Biotechnology: Status and Uses

Animal Science 434

John J. Parrish

Animal Science 434

John J. Parrish

Reproductive Biotechnology'sReproductive 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

In 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 Development

As a direct result of research conducted by Parrish, Susko-Parrish, First, Leibfried-Rutledge, Critser and Ax, more than 450,000 IVP embryos are produced world wide each year and the procedures are the among the most cited publications in all of reproductive physiology!

In 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– Humans

Stimulating Follicular DevelopmentStimulating Follicular Development

Est

rus

Est

rus

eCG orFSH

eCG orFSH

Progesterone From C.L.

Progesterone From C.L.

First FollicularWave

First FollicularWave

10-1210-12

MultipleOvulations

MultipleOvulations

PGF2PGF2

Non-Surgical Embryo Flush

Non-Surgical Embryo Flush

Day 6 or 7Day 6 or 7

Stage 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)

ET - Scheme

ET - Scheme

Embryo 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 DNA

Hy-AntigenHy-Antigen

Octopus Springs - Yellowstone National ParkOctopus Springs - Yellowstone National Park

Home of Thermus aquaticus - Taq PolymeraseHome of Thermus aquaticus - Taq Polymerase

PCR Approach to Embryo SexingPCR Approach to Embryo Sexing

XY

F M F F F F F F

Sperm Gender SelectionSperm Gender Selection

Selection of X or Y spermSelection of X or Y sperm

PERCENT DNA DIFFERENCES BETWEEN X and Y CHROMOSOME

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!!!X Chromosome has more DNA!!!

++ --+-

LLAASSEERR

90°0°

XX ssoorrttYY ssoorrtt

SpermStained

With DNASensitive

FluorescentDye

SpermStained

With DNASensitive

FluorescentDye

Flow Cytometer Separation of X and Y SpermFlow Cytometer Separation of X and Y Sperm

Sperm Gender SelectionSperm 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

Fetal SexingFetal Sexing

OR

Ultrasound EvaluationUltrasound Evaluation• Day 55 - 65• Day 55 - 65

CloningCloning

Split morulaSplit morula

Cloning by Nuclear TransferCloning by Nuclear Transfer

Cycles are limited

Only 3 - 4 cycles

Nuclear Cloning to Produce Stem CellsNuclear Cloning to Produce Stem Cells

Cells of InterestGrown in Culture

Dish

Cells of InterestGrown in Culture

Dish

Mature Oocyte enucleated and cell

of interest fused

Mature Oocyte enucleated and cell

of interest fused

Embyro??Develops

Embyro??Develops

Inner Cell Mass Cells isolated and grown in culture

Inner Cell Mass Cells isolated and grown in culture

DifferentiateDifferentiatePotential Tissue:• Neural• Adipose• Connective• Heart

Potential Tissue:• Neural• Adipose• Connective• Heart

Stem Cells Grown in Culture and Allowed to Differentiate

Stem Cells Grown in Culture and Allowed to Differentiate

http://www.ansci.wisc.edu/jjp1/as434/powerpoint/fa07/stem_cells.mov

Benefits of Stem Cell Research

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

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

Genetic Manipulation

Genetic Manipulation

Transgenic for Growth Hormone

Transgenic for Growth Hormone

Gene Transfer Using Micro-Injection of Pronuclei

Gene Transfer Using Micro-Injection of Pronuclei

Less than 1% efficiency

Gene Transfer

Using Viral Transfection

Gene Transfer

Using Viral Transfection

Better success but left with potential for viral replicationBetter success but left with potential for viral replication