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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

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

  • Oocyte isolation and maturation

  • Sperm preparation

  • Sperm capacitation

  • Fertilization

  • Embryo Development


Oocyte Aspiration


Searching

Isolation


Culture

24 Hours Incubation

  • Maturation Medium

  • Tyrode’s

  • FSH, LH, Estradiol

  • Fetal Calf Serum

39°C, 5% CO2 in Air


Cumulus Expansion

Unexpanded

Expanded


Sperm Preparation

Semen

45% Percoll

90% Percoll


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


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


Stimulating Follicular Development

  • eCG (PMSG)

    • Single injection

  • FSH

    • 8 injections

    • AM/PM

    • Decreasing doses


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


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


Embryo Recovery

  • 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


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%)


ET - Scheme


Embryo Transfer Uses

  • 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


Octopus Springs - Yellowstone National Park

Home of Thermus aquaticus - Taq Polymerase


PCR Approach to Embryo Sexing

Y

X

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

X Chromosome has more DNA!!!


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


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


Fetal Sexing

OR

Ultrasound Evaluation

  • Day 55 - 65


Cloning

Split morula


Cloning by Nuclear Transfer

Cycles are limited

Only 3 - 4 cycles


First Clones by Nuclear Transfer at the UW


Cloning Uses

  • Production of identicals

    • Agricultural uses

    • Transgenics

    • Stem cell production and research


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


Potential Uses of Genetic Manipulation

  • 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


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