IVF and Embryo Transfer in Livestock

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Questions and Answers

What is the primary purpose of using In Vitro Fertilization (IVF)?

To store semen indefinitely

To diagnose genetic disorders in embryos

To assist when artificial reproductive techniques fail (correct)

To fertilize sperm and egg inside the body

What is the first step in the IVF process?

In Vitro Fertilization

Ovarian stimulation (correct)

Egg retrieval

Pre-implantation Genetic Diagnosis

Which of the following animals has not been reported to have IVF offspring?

Elephants (correct)

Squirrels

Pigs

Cats

What technique is utilized to help with embryo diagnosis before transfer in IVF?

Pre-implantation Genetic Diagnosis (PGD) Signup and view all the answers

In which environment does In Vitro Fertilization occur?

In vitro (outside the animal's body) Signup and view all the answers

What is the main purpose of embryo transfer in livestock?

To improve the reproductive rate of selected females. Signup and view all the answers

What is done to the embryos before they are transferred to surrogate mothers?

They are flushed, examined, and evaluated for number and quality. Signup and view all the answers

Which of the following describes a transgenic animal?

An animal that contains genes from different species inserted into its genome. Signup and view all the answers

How does embryo transfer reduce disease transfer?

By ensuring only selected embryos are used for transfer. Signup and view all the answers

What is achieved by superovulating selected females?

Increased number of viable offspring from fewer inseminations. Signup and view all the answers

What is the role of the promoter in the construction of a transgene?

To control the expression of the gene Signup and view all the answers

Which step follows the microinjection of the transgene into a fertilized egg?

Implantation into a surrogate mother Signup and view all the answers

What is a characteristic feature of embryonic stem (ES) cells?

They can renew themselves and differentiate into various specialized cell types Signup and view all the answers

Which method is NOT commonly used to incorporate a transgene into embryonic stem cells?

Chemical transformation Signup and view all the answers

What is the main outcome of using the microinjection method in selective breeding?

To create transgenic animals with desired traits Signup and view all the answers

What happens to cells with the desired genetic alteration after DNA-mediated gene transfer in embryonic stem cells?

They are cultured and selectively grown Signup and view all the answers

How are genetic mosaics typically produced in transgenic animal development?

By inserting altered embryonic stem cells into blastocysts Signup and view all the answers

Which component of the transgene marks the end of transcription?

Terminator Signup and view all the answers

What is the purpose of the retro virus in the embryonic stem cell method?

To introduce the transgene into the ES cells Signup and view all the answers

Which of the following best describes adult stem cells?

They primarily serve as a repair system for the body Signup and view all the answers

What is the function of glycerol in semen storage?

Prevents ice crystal formation Signup and view all the answers

Which of the following steps is essential for ovarian stimulation in IVF?

Administration of hormones Signup and view all the answers

What is a potential reason for utilizing IVF in reproduction?

To address blocked reproductive systems Signup and view all the answers

What is the purpose of Pre-implantation Genetic Diagnosis (PGD) in IVF?

To evaluate embryo viability before transfer Signup and view all the answers

What is the primary benefit of using embryo transfer (ET) in livestock production?

It allows for the propagation of genetic stocks from superior females. Signup and view all the answers

Which of the following describes the procedure performed on embryos before their transfer to a surrogate mother?

They are flushed from the uterus and examined for quality. Signup and view all the answers

What is an essential step in producing transgenic animals?

Introducing a gene from a different species into the animal's genome. Signup and view all the answers

What advantage does superovulation provide in the embryo transfer process?

It increases the quantity of embryos available for transfer. Signup and view all the answers

Which of the following is NOT a characteristic of embryonic stem (ES) cells?

They are derived exclusively from adult tissues. Signup and view all the answers

What is the primary role of the termination sequence in a transgene?

To mark the end of transcription Signup and view all the answers

Which method allows for the incorporation of a transgene into embryonic stem cells?

Electroporation Signup and view all the answers

What type of cells are embryonic stem cells able to differentiate into?

All specialized embryonic tissues Signup and view all the answers

During the microinjection method, where is the transgene injected?

Into the male pronucleus of a fertilized egg Signup and view all the answers

Which of the following describes the function of a promoter in a transgene?

To control gene expression and initiation of transcription Signup and view all the answers

What is typically done to the fertilized eggs before implanting them into a pseudo-pregnant female?

They are incubated overnight to develop to a 2-cell stage Signup and view all the answers

Which type of stem cells is primarily responsible for the repair system in adult tissues?

Adult stem cells Signup and view all the answers

What is the primary outcome after successfully creating a transgenic animal from embryonic stem cells?

It is usually a genetic chimera Signup and view all the answers

What does the term 'genetic mosaic mice' refer to?

Mice that have multiple origins of genetic material in their tissues Signup and view all the answers

Study Notes

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.

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Description

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.