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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 l.jpg

Biotechnology: Status and Uses

Animal Science 434

John J. Parrish


Reproductive biotechnology s l.jpg

Reproductive Biotechnology's

  • Artificial insemination

  • In vitro embryo production

  • In vivo embryo production

  • Embryo transfer

  • Gender selection

  • Genetic engineering

  • Cloning


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In Vitro Production of Embryos

  • Oocyte isolation and maturation

  • Sperm preparation

  • Sperm capacitation

  • Fertilization

  • Embryo Development


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Oocyte Aspiration


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Searching

Isolation


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Culture

24 Hours Incubation

  • Maturation Medium

  • Tyrode’s

  • FSH, LH, Estradiol

  • Fetal Calf Serum

39°C, 5% CO2 in Air


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Cumulus Expansion

Unexpanded

Expanded


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Sperm Preparation

Semen

45% Percoll

90% Percoll


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Fertilization


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Embryo Development

2-Cell

4-Cell

8-Cell

Morula


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Embryo Development

Blastocyst


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In Vivo Embryo Production

  • Normal cycling female

    • horses

  • Superovulated female

    • Cattle

    • Sheep

    • Goats

    • Deer

    • Humans


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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


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Stimulating Follicular Development

  • eCG (PMSG)

    • Single injection

  • FSH

    • 8 injections

    • AM/PM

    • Decreasing doses


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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


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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


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Embryo Recovery

  • Early to mid blastocyst

    • Day 6 to 7 (estrus, breeding = day 0)

  • Flush uterus

    • Surgical

    • Nonsurgical


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Procedure for Non-Surgical Embryo Flush


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Non-Surgical Embryo Flush


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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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Embryo Transfer

  • Recipient must be synchronized with donor or 1 day behind

  • Surgical

    • Flank

  • Nonsurgical

    • Similar to AI but going through diestrus cervix


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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%)


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ET - Scheme


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Embryo Transfer Uses

  • Introduction of new genetics

  • Import/Export

  • Twinning

  • Coupling with other biotechnologies


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Embryo Sexing

  • 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

Y

X

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

X Chromosome has more DNA!!!


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Flow Cytometer Separation of X and Y Sperm

Sperm

Stained

With DNA

Sensitive

Fluorescent

Dye

9

L

A

S

E

R

-

+

+

-

Y

s

o

r

t

X

s

o

r

t


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Flow Cytometer for Sperm Separation


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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


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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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First Clones by Nuclear Transfer at the UW


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Cloning Uses

  • Production of identicals

    • Agricultural uses

    • Transgenics

    • Stem cell production and research


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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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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


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Potential Uses of Genetic Manipulation

  • Production of spare body parts

  • Disease resistance

  • Increased production traits


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Gender Selected Semen


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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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