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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

Biotechnology: Status and Uses

Animal Science 434

John J. Parrish

reproductive biotechnology s
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
In Vitro Production of Embryos
  • Oocyte isolation and maturation
  • Sperm preparation
  • Sperm capacitation
  • Fertilization
  • Embryo Development
searching
Searching

Isolation

culture
Culture

24 Hours Incubation

  • Maturation Medium
  • Tyrode’s
  • FSH, LH, Estradiol
  • Fetal Calf Serum

39°C, 5% CO2 in Air

cumulus expansion
Cumulus Expansion

Unexpanded

Expanded

sperm preparation
Sperm Preparation

Semen

45% Percoll

90% Percoll

embryo development
Embryo Development

2-Cell

4-Cell

8-Cell

Morula

in vivo embryo production
In Vivo Embryo Production
  • Normal cycling female
    • horses
  • Superovulated female
    • Cattle
    • Sheep
    • Goats
    • Deer
    • Humans
stimulating follicular development

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 development14
Stimulating Follicular Development
  • eCG (PMSG)
    • Single injection
  • FSH
    • 8 injections
    • AM/PM
    • Decreasing doses
stimulating follicular development15

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

stimulating follicular development16

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
Embryo Recovery
  • Early to mid blastocyst
    • Day 6 to 7 (estrus, breeding = day 0)
  • Flush uterus
    • Surgical
    • Nonsurgical
stage of embryo at recovery
Stage of Embryo at Recovery

Tight Morula (day 5 - 7)

Early Blastocyst (day 7 - 8)

Blastocyst (day 7 - 9)

embryo transfer
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
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%)
embryo transfer uses
Embryo Transfer Uses
  • Introduction of new genetics
  • Import/Export
  • Twinning
  • Coupling with other biotechnologies
embryo sexing
Embryo Sexing
  • Hy Antigen
    • Associated with male cells
  • PCR and Detection of Y and X DNA
slide27

Octopus Springs - Yellowstone National Park

Home of Thermus aquaticus - Taq Polymerase

sperm gender selection
Sperm Gender Selection

Selection 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

X Chromosome has more DNA!!!

slide31

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

sperm gender selection33
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
Fetal Sexing

OR

Ultrasound Evaluation

  • Day 55 - 65
cloning
Cloning

Split morula

cloning by nuclear transfer
Cloning by Nuclear Transfer

Cycles are limited

Only 3 - 4 cycles

cloning uses
Cloning Uses
  • Production of identicals
    • Agricultural uses
    • Transgenics
    • Stem cell production and research
gene transfer using viral transfection
Gene Transfer Using Viral Transfection

Better success but left with potential for viral replication

problems with gene transfer
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
Potential Uses of Genetic Manipulation
  • Production of spare body parts
  • Disease resistance
  • Increased production traits
dairy production in the tropics
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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