Biotechnology: Status and Uses

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Biotechnology Status and Uses

• Animal Science 434
• John J. Parrish

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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
• Tyrodes
• FSH, LH, Estradiol
• Fetal Calf Serum

39C, 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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Stimulating Follicular Development
Multiple Ovulations
eCGorFSH
Ovulation
Estrus
Estrus
Progesterone From C.L.
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Stimulating Follicular Development

• eCG (PMSG)
• Single injection
• FSH
• 8 injections
• AM/PM
• Decreasing doses

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Stimulating Follicular Development
AI at 12 and 24 hr after coming into estrus
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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
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0
0
L
A
S
E
R
-


-
X

s
o
r
t
Y

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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