IVF and Embryo Transfer in Livestock

Food Biotechnology: Animal Biotechnology

• Modern techniques for improving animal lines have been developed over the past 40 years
• Artificial insemination (in vivo fertilization) significantly impacts the dairy industry
• In vitro fertilization, embryo cloning, and nuclear transplantation are becoming more common
• Transgenic technology provides valuable therapeutic proteins and disease-resistant strains, as well as lean meat/efficient growth strains

GMO Animals

• GMO animals can be divided into six categories:
  • Investigating human diseases
  • Producing industrial or consumer products
  • Producing human therapeutic proteins
  • Enhancing animal interactions with humans (e.g. hypoallergenic pets)
  • Enhancing food quality traits (faster growth, efficient digestion)
  • Improving animal health (disease resistance)

Examples of GMO Animals

• Annie, a purebred Jersey calf, produced lysostaphin (a bacterial gene) in 2000
• Genetically engineered and cloned pigs are used for human organ transplants

Animal Biotechnology Areas

• Biotechnology in animal breeding
• Transgenic animals
• Animal cloning
• Transgenic animal cell culture / bioreactor
• Genome editing

Biotechnology in Animal Breeding

• Goal: Enhance quality and quantity of animal products (meat, milk, eggs, wool)
• Traditional breeding (selective breeding): Mating selected animals based on phenotypic criteria
• Modern animal breeding using biotechnology
  • Artificial insemination (AI)
  • In vitro fertilization (IVF)
  • Embryo transfer (ET)
  • Genetic maps and gene marker-assisted breeding

Artificial Insemination (AI)

• A process where sperm is introduced into a female reproductive tract without sexual intercourse
• First AI station for cattle breeding established in Germany in 1942
• Cost-effective and enables selecting high-value male animals
• More than 80% of cows in industrialized countries become pregnant through AI

Advantages of AI

• Genetic improvement: Access to superior genetics from geographically distant or expensive bulls improves the herd's quality
• Disease control: Reduces sexually transmitted disease risk among livestock
• Increased efficiency: Reduces the number of bulls needed per farm, leading to cost savings

Artificial Insemination Steps

• Semen collection using artificial vaginas (often commercially available)
• Semen storage: Long-term storage using liquid Nitrogen with glycerol
• Insemination: Recto-vaginal method using a sterilized AI gum inseminator

In Vitro Fertilization (IVF)

• Used when artificial reproductive techniques fail, including blocked reproductive systems, unresponsive ovaries, poor semen quality, and diseases.
• Fertilization occurs outside the animal's body (in vitro)
• Advances in embryo production and cryopreservation have enabled IVF in numerous species (mice, rats, hamsters, cats, guinea pigs, squirrels, pigs, cows, monkeys, and humans).

Steps in IVF

• Ovarian stimulation: Inducing superovulation with hormones
• Egg retrieval
• In-vitro fertilization (fertilizing the eggs with sperm outside the body)
• Embryo selection: Choosing the best embryos using Pre-implantation genetic diagnosis (PGD)
• Embryo transfer: Transferring selected embryos into the uterus of a surrogate mother.

Transgenic Animals

• Animals with one or more genes from another source (e.g., human or microorganisms) or altered genes
• "Supermouse": Early 1980s example that grew much larger due to a human growth hormone gene insertion.
• Fostered expectation of quicker growth hormone boosted levels for farm animals.
• GMO animals can be used for research to understand disease pathways and human diseases
• Example includes the "Beltsville Pig"

Transgenic Animals (Problems)

• Numerous attempts in creating transgenic animals to improve leaner meat, better feed efficiency and faster growth
• Skeleto abnormalities are found in transgenic animals.
• Problems with gene expression are likely linked to the increased expression of growth hormone.
• Example includes the "Beltsville Pig," which showed increased growth rate, reduced fat, and improved feed efficiency, but developed unforeseen health problems.

The Infamous "Beltsville Pig" (1989)

• Researchers genetically modified pigs to include a human growth hormone gene.
• Initially successful, increasing growth rates, reducing fat, increasing feed efficiency
• Unforeseen problems with health (kidneys, liver, uncoordinated movements, bulging eyes, ulcers, heart issues and pneumonia).

Basic Procedure for Production of Transgenic Animals

• Identifying a target gene
• Cloning the gene
• Constructing a gene suitable for transfer
  • Most important step
  • Embryonic stem cell method, is the first method
  • Microinjection method
• Checking integration of the foreign gene in the animal's genome
• Checking if the foreign gene is expressed to produce the desired proteins or the wanted effects
• Checking transmission of the trait (e.g., through inheritance breeding)
• Selective breeding

Methodology: Transgene Construction

• The transgene should comprise a promoter, a gene of interest, and a terminator sequence.
• The promoter region activates the expressed gene for expression in the new organism
• The terminator segment marks the section where the transgene expression stops.

Methodology: Introducing the Foreign Gene into Animal

• Microinjection method for introducing foreign genes into fertilized eggs
• Embryonic stem cell method involves culturing embryonic stem cells with the foreign gene to create a chimera. Then, combining the chimera with an embryo to produce an animal carrying the genetic modification.
• Retroviral method uses retroviruses to insert the foreign gene into the animal's DNA.

Microinjection Method

• Superovulating a female animal to collect eggs
• Fertilizing eggs in vitro.
• Injecting foreign genes into the male pronucleus of fertilized eggs
• Incubating fertilized eggs overnight and to grow into 2-cell stage.
• Positioning the embryo into a pseudopregnant female
• Cloning and producing more embryos carrying the genetic modification

Microinjection Method -- Increase the Yield of Milk

• Microinjecting the recombinant bovine somatotropin (rBST) gene into the pronucleus.
• Implanting the egg into a pseudopregnant cow.
• Breeding the transgenic cow.
• Increasing milk yield by ~20%.

Stem Cells

• Stem cells can divide and differentiate into various specialized cell types.
• Embryonic stem cells are derived from the inner cell mass of blastocysts and can differentiate into all specialized tissues.
• Adult stem cells function as a repair system by replacing specialized cells in body tissues such as blood, skin, and intestines

Embryonic Stem Cell Method

• Creating transgenic animals through manipulating embryonic stem cells (ES cells).
• Incorporating the transgene into the ES cells using microinjection, retroviruses, or electroporation.
• Growing the transgenic stem cells in vitro and inserting them into blastocysts.
• Implanting the blastocysts into a surrogate mother.

Retroviral Method

• Retroviruses can infect the early embryos and insert proviral DNA copies of their genomes into the mouse chromosome
• Exposing embryos to the medium containing recombinant retroviruses
• Gene expression efficiency is limited in viral constructs

CRISPR-Cas9

• A commonly used genome editing technique adapted from a naturally occurring prokaryotic genome editing system in bacteria
• The bacteria capture DNA snippets from invading viruses and form CRISPR arrays
• If re-attacked, the bacteria produces RNA segments from their CRISPR arrays to target virus DNA.
• The bacteria use Cas9 (or other similar enzymes) to cut the DNA apart, thus disabling the virus

CRISPR- Working Principle

• Stage 1 (foreign DNA acquisition)
• Stage 2 (CRISPR RNA processing)
• Stage 3 (RNA-guided targeting of viral elements)
• Palindromic sequence - sequence in one strand that matches the sequence of the complementary strand when read in the same direction.

CRISPR-Cas9 Introduces a Break at Target Location

• A CRISPR guide RNA (gRNA) complex with Cas9 direct where to cleave the targeted DNA, guided by complementary sequences.
• The cell tries to repair the DNA break by causing insertion or deletion of nucleotides, which leads to changes in the reading frame and premature stop codon.

Application of CRISPR... and more to come

• Genome/gene editing techniques, for example CRISPR, could be applied to diverse fields like drug development, animal models, gene variation materials, and more.

Genome Editing in Disease-Resistant Animals

• Many animal viruses are difficult to control using conventional methods such as vaccination
• Transgenic technology may provide an efficient approach to control viruses.
• By altering viral receptors, animals may become resistant to certain infections.
• Some genome editing cases are successful in preventing viruses from attaching to animal cells, making them resistant to infections
• Example includes PRRS resistant pigs

Example: PRRS Resistant Pigs

• Porcine Reproductive and Respiratory Syndrome Virus (PRRSV): A highly prevalent and costly virus for pigs.
• Using CRISPR-Cas9, a tiny region of pig DNA is deleted, preventing the PRRS virus from attaching to pig cells.
• Tests showed gene-edited pigs resisted PRRSV infection without other negative consequences.

Transgenic Animals: Potential Benefits and Concerns

• Potential benefits: specificity, speed of breeding, flexibility (e.g., cross species traits), and economy (e.g., feed efficiency, fewer treatments)
• Animal welfare concerns: transgenes upsetting genome expression, virus transfer, dissemination, and food safety concerns.

Summary: Transgenic Animals

• Technologies for creating transgenic animals and gene regulation are advancing rapidly
• Transgenic animals play a significant role in studying gene function, human diseases, and biomedical applications.
• Progress in transgenic farm animals has been limited by public resistance to genetically modified foods.
• Transgenic fish were the first GM food animals in the food supply, not the last.

This quiz explores the key concepts of In Vitro Fertilization (IVF) and embryo transfer specifically in livestock. Participants will learn about the purposes, processes, and techniques related to these biotechnological advancements in agriculture and animal breeding.