Biotechnology: Status and Uses

Selected Images from Lecture

Reproductive Biotechnology's

• Artificial insemination
• In vitro embryo production
• In vivo embryo production
• Embryo transfer
• Gender selection
• Genetic engineering
• Cloning

Artificial Insemination

In Vitro Production of Embryos

• Oocyte isolation and maturation
• Sperm preparation
• Sperm capacitation
• Fertilization
• Embryo Development
  

In Vivo Embryo Production

• Normal cycling female
  • horses
• Superovulated female
  • Cattle
  • Sheep
  • Goats
  • Deer
  • Humans

Stimulating Follicular Development

• within a normal cycle
  • eCG or FSH given on day 17
• a cycle shortened with PGF
  • give eCG or FSH on day 10-12
  • PGF two days later

Stimulating Follicular Development

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

Stimulating Follicular Development with eCG

• eCG given day 0
• PGF given AM and PM of day 2
• estrus day 3.5 - 5
• breed 12 and 24 hrs after start of estrus

Stimulating Follicular Development with FSH

• day 0 -3.5 give FSH injection both AM and PM of each day
  • use decreasing doses
• PGF given AM and PM of day 2
• estrus day 3.5 - 5
• breed 12 and 24 hrs after start of estrus

Embryo Recovery

• Early to mid blastocyst
  • Day 6 to 7 (estrus, breeding = day 0)
• Flush uterus
  • Surgical
  • Nonsurgical

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%)

Embryo Transfer Uses

• Introduction of new genetics
• Import/Export
• Twinning
• Coupling with other biotechnologies
  

Gender Selection

• Embryo Sexing
  • Hy Antigen
    • Associated with male cells
  • PCR and Detection of Y and X DNA
• Sperm Selection
  • Differences in DNA between X and Y chromosome
    • X has more DNA
    • Species differences
      • Human 2.9%
      • Cattle 3.8%
      • Chinchilla 7.5%
      • Turkey 0%
  • Flow cytometer used to separate X and Y sperm based on DNA differences
    • Only method that works!
    • Very few sperm recovered
    • Currently not suitable for AI use
• Fetal Sexing
  • ultrasound at day 55 - 65 of gestation
  • what do you do with unwanted sex fetus

Application of Biotechnology to Replace Superovulation and Embryo Transfer (single calf heifer project)

• Follicular aspiration (replace superovulation), IVF with gender selected semen (optional), In vitro culture of embryo, embryo sex evaluation (not needed if gender selected semen), embryo transfer
  • recipient is heifer (13 to 15 mon of age)
• Save calf and market mother
  • young enough to grade choice
  • problem though in could not get grader's to go along with this classification
  • bones change after a pregnancy
• System requires all female calves and some twinning to be sustaining
  • always some death loss

Crossbreeding

• Application of Biotechnology to Beef Production
  • Follicular aspiration (replace superovulation), IVF with semen from another breed, In vitro culture of embryo, embryo transfer
  • possibly include twinning (multiple embryo transfer) if use gender selection
• 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

Cloning

• Splitting of morula
• Nuclear transfer
  • Example of clones done at University of Wisconsin
• Cloning Uses
  • Production of identicals
    • Agricultural uses
    • Transgenics

Cloning by Nuclear Transfer

• allows selection and multiplication of a superior embryo
• limited number of cycles
  • 3 - 4
• Types of Cells that can be Cloned
• Embryos
• Somatic Cells
  • Mammary epithelial cells
  • Cumulus cells
  • Fetal cells

Stem Cells

• Regenerative medicine
• drug toxicology
• mechanisms of human disease and development

Genetic Manipulation

• Methodology
• Micro-Injection of Pronuclei
• Viral Transfection
  

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
  

Review Quizzes