ADDIS ABABA UNIVERSITY

COLLEGE OF VETERINARY MEDICINE & AGRICULTURE

Department of Clinical Studies

Tefera Yilma (PhD)

Course Title: Theriogenology II
(Vetm4162)
Sub-topic: Assisted Reproductive
Technology(ART)-Embryo Technologie
April, 2024
Outline
Assisted Reproductive Technology (ART) – Embryo
technology (biology of the ovum)
 Multiple Ovulation and Embryo Transfer (MOET)
 In Vitro Embryo Production (IVEP)
 Ova collection, in vitro maturation,
in vitro fertilization, in vitro culture
 Micromanipulation of gametes &
embryos – Semen - / Embryo Sexing
 Cloning
 Production of transgenic animals - Stem cells
Introduction
Embryo technologies (biology of the ovum) - various
applications
Assisted Reproductive Technologies
(human, animals)
• Control of Fertility
• Preservation of diversity
through reproduction of
endangered species / breeds
...  prone to extinction!
• Genetic Selection (animals)
Very use full…

 A lot of interests!
 More genetic progress (selection schemes): gene
editing, embryo sexing, and cloning techniques can
enhance genetic progress and precision in cattle
breeding
 Commercial (farmers, multiplication of best females)
 International genetic exchange - Exchange of
genetic material (safe)
But…
 A lot of questions???
• Ethical maters (+/-, techniques and species)
Multiple Ovulation and Embryo Transfer (MOET)

Multiple ovulation/superovulation: Induction of multiple ovulations

by ovarian super stimulation using fertility drugs (gonadotrophins)

Using PMSG/eCG, FSH to induce multiple ovulations in the ovaries of

the donor animal  to increase supply of embryos from dams of superior
genetic merit

… aims at the use of genetically superior dams

―Superovulation‖-ovarian response varies with:
Dosage regime
Types of gonadortopins
Status of follicular development … more ova, if large number of
follicles are present at time of treatment
MOET (cont’d)

Embryo transfer…
ET involves
Collection of embryo(s) from a donor
animal (6 to 8 days after service )
…Embryos flushed (donor animals)

Placement into the uterus of a recipient

… Embryos transferred (recipient animals)

 MOET…  to amplify reproductive rates

of valuable females - fivefold or more per
lifetime
•Scarcity /endangered
•proven genetic value
•unique characteristics- disease resistance
MOET (cont’d)

Advantages
•number of calves from genetically
superior cows (females of superior genetic
merit)
•marketing opportunity: sale of offspring,
pregnancies and embryos
•to extend the productivity of valuable dams
•to extend the productive life of females
(injured/or can not develop a young but
are still fertile)
•to accelerate genetic progress  more rapid
proof of a dam through greater number of
offspring in a short period of time
MOET (cont’d)

Disadvantages/Limitations
 High cost of superovulation -
ET program
 Requires a high level of
management
 Not all donors respond to
the superovulation treatment
DONNOR
RECIPIENTS
Superovulation Estrus synchronization

AI - - - - Oestrus

Flush(D7) Transfer (D7)

MOET (cont’d)

Gonadotrophins:
A) Multiple ovulations using PMSG (eCG): I.M injection of 2000–
2500 IU
Advantage of using eCG):
•A single injection b/c eCG has a longer biological half-life than FSH
•Prostoglandin (48-72 hours later)
•regression of the CL
•induce estrus: 40 – 56 hours later
•GnRH or hCG to induce ovulation

Limitations (eCG/ PMSG):

•eCG effect persists after induced estrus- long biological half-life
(eCG-antiserum to overcome the persistent effect)
•embryo transport adversely affected
•poor recovery rate of embryos
•Expensive
MOET (cont’d)

B) Follicle Stimulating Hormone (FSH)

Shorter half-life after i.m. injection of only five

hours (Demoustier et al. 1988) twice injection
daily (mornings and evenings) for 4 days with
decreasing doses
 Advantage (FSH)
• better superovulatory response than eCG
…PGF2 administration 48–72 hours after
initiation of treatment with FSH – to induce estrus

Limitation: repeated injection (twice/day for 4 days)

 Folliculogé
Folliculogénèse

Hormone(s)
Folliculogé
Folliculogénèse
peu active trè
très active

Reference FSH
FSH
Heat AI TRANSFERT
FLUSH

0 D9-D13 0 D7

PGF2

FSH: 500 µg FSH (STIMUFOL , Follitropin …. ND)

8 im injections decreasing doses at 12 hrs interval
Prostaglandins: 0.5 - 1 mg cloprostenol at 5th FSH injection
 FSH decreasing doses for superovulation of cattle
Bos Taurus Bos Indicus Heifers

AM PM AM PM AM PM

Day 1 5 mg 5 mg 4 mg 4 mg 3 mg 3 mg

Day 2 4 mg 4 mg 3 mg 3 mg 3 mg 3 mg

Day 3 3 mg 3 mg 2 mg 2 mg 2 mg 2 mg

Day 4 2 mg 2 mg 2 mg 2 mg 2 mg 2 mg

Total 28 mg 22 mg 20 mg
MOET (cont’d)

Steps in embryo transfer

1. Selection of donors and sires

Two broad criteria:
a) Genetic superiority
•milk production (yield), milk composition
•growth rates, calving ease
•disease resistance
b) Commercial - likelihood of producing large numbers of usable
embryos
MOET (cont’d)
2. Selection and preparation of recipient
animals
…The condition and preparation of the recipients influence the
pregnancy rate (success rate) after embryo transfer:

•good breeders
•sexually mature, cycling, three months post parturition
•good state of nutrition
•healthy animals - free form brucellosis, trichomoniasis, and
other genital tract diseases
MOET (cont’d)

3. Synchronization of estrus between donor and

recipients
Synchronization b/n the stage
of ovum and the reproductive
tract, … recipients should be in
estrus at the same time as the
donor
1. PGF2 or a suitable analogue
during the luteal phase (common)
2. Progestin method
MOET (cont’d)

4. Estrus detection

5. Insemination (donor)
AI:- based on first observed estrus

AI … more often, with more sperm per

insemination of superovulated than other donors:
•multiple follicles ovulate over a period of time
•transport of sperm and ova is altered/affected by
superovulatory treatment
MOET (cont’d)
6. Collection of embryos
Surgically or non-surgically
(intact animal) but also from the
oviducts or uteri of slaughtered
animals

•Embryos recovered 6 to 8 days

after service (day 0)
….embryos enclosed with
zona pellucida
Collection: at late morula
or blastula stage of development
MOET (cont’d)

Non-surgical
recovery (common)

Using a two or three-way

flushing system (catheters):
…one passage for air and one/
two for fluid

Each uterine horn is filled

With 40 to 60ml of flushing
medium with foley catheter-
repeated until 400 to 800ml
of medium have been used
 MOET (cont’d)

Non-Surgical Flushing and Recovery of Embryos

-1978- -2010-
 MOET (cont’d)

Surgical methods
Laparotomy to expose
the reproductive tract
Either from the fimbria
toward the uterus or from
the uterotubal junction
toward the fimbria
•flushing medium
•a syringe and blunt
needle
•a small glass tube
for collecting flushing,
medium inserted in to the
infundibulum
MOET (cont’d)
MOET (cont’d)

7. Selection of embryo
for transfer
Isolation and
Classification under a
microscope
Morphologic examination
Embryos are classified as:
Excellent, Good, Fair or Poor
in quality
 MOET (cont’d)
Embryo Grading
Criteria for classifying Embryo Quality
embryos 1 Excellent: spherical, symmetrical
with cells of uniform size
-even number of cells
2 – Good: few extruded blastomeres,
-uniform division irregular shape
3 – Fair: extruded blastomeres, few
-healthy zona pellucida degenerated cells
4 – Poor: Numerous extruded
blastomeres, degenerated cells

… Only morphologically normal embryos are

transferred to the recipient animals
MOET (cont’d)
MOET (cont’d)

Embryos: generally spherical or ovoid, not too light

nor too dark in color, uniform cell size
Deviations from normal include:
•irregular cell sizes, blastomeres of variable non-uniform size
•large vacuoles in cells
•areas of degeneration in the embryos, fragmentation of
cytoplasmic and nuclear material

•cellular debris in the morula

•abnormal shape of morula or blastocyst
•cells which are not compacted with the main cell mass
(extruded blastomeres), and a damaged zona pellucida
MOET (cont’d)

7 -8 days after
fertilization

Freezing Transfer to
recipients
MOET (cont’d)

Loading of a Straw for Freezing

MOET (cont’d)

8. Transfer of embryo

Non- Surgical transfer

•more common
•…depositing of embryos in the
uterus through the cervix
with an AI straw gun
Transfer of embryo (cont’d)

Surgical transfer: Flank laparotomy under local anesthetic is

used in cattle:
1. Tip of a capillary pipette
containing the embryos is
inserted into the imfundibulum
and ampulla of the oviduct … 
embryos are deposited in a
drop or two of medium

2. Wall of the uterine horn is

punctured with a blunt needle,
the embryos are expelled from
the tip of the capillary pipette
inserted in to the uterine lumen
Number of flushes (bovine) in 2009

60000 52921

50000
Africa
40000 Asia (Japan)
Europe
30000
N.America
20000 16856 S.America
12065
10924 10070 Oceania
10000
1446
0
 In Vitro Embryo Production: – In
Vitro Fertilization (IVF):
Oocyte collection, evaluation, in vitro maturation,
and in vitro fertilization (in the lab)
 Oocytes collection méthodes /sources:
1) Slaughterhouse ovaries - Aspirate the eggs (syringe-niddle)
2) Ovum pick up (cow)
(Oocytes are recovered from the ovary via
ultrasound-guided needle through the 1 = Oocyte
2 = Pellucid zone
vagina-‖ovum pick-up”) 3 = Stratum granulosum
4 = Theca interna
5 = Theca externa
3) Endoscopy (human) 6 = Antral follicle
7 = Cumulus oophorus (Granulosa cells, together with the oocyte)
8 = Basal lamina between theca and stratum granulosum
 Collection of oocytes in vivo

In vitro Maturation  in vitro fertilization  Morula/ Blastocyst stage

Transfer to in vivo
 History of IVF
• Rabbit (1954)
• Golden hamster (1963)

•IVM of oocytes from

various species (1965-1988)

• Human (1969 1978)

 2010
The Nobel Medal for
Physiology or Medicine
Inventas vitam juvat excoluisse
per artes

"And they who bettered life on

earth by their newly found
mastery“

Robert G. Edwards

“For the development of in vitro fertilization"

 In vivo  in vitro...
How do we simulate
this in the laboratory?
2 Sources of oocytes:
 Slaughterhouse ovaries
Ovum pick up (ultrasoud guided
ovum pick up – OPU)
Collect ovaries Aspirate the eggs
(slaughter (oocytes)
house) from the ovaries
Ovum pick up (cow)
In-vitro fertilization (IVF) (cont’d)

Ovum pick up IVF:

Ovaries aspirated once/twice weekly from cyclic cows/heifers
Ovaries aspirated weekly up to day 100 of pregnancy
• Selected oocytes are incubated for 24 - 48 hours – Maturation!
• Oocytes and sperm co-culture for 6 -24 hours– Fertilization!
• Embryos cultured for 6 – 7/8 days
(in vitro culture!)
…Morula / Blastocyst for transfer
Rate of success
Oocyte aspirated of intact ovaries:
•50% of oocytes are abnormal, discarded
•50% used for IVF
 75% of these are fertilized and used for ET
 50% overall pregnancy rate!!
In-vitro fertilization (IVF) (cont’d)

Advantages OPU for IVF

 Production of large number of embryos
 Not necessary to super ovulate the donor cows, no exposure to
hormones
 Not necessary to synchronize estrus (donor & recipients)
 Production of sexed embryos- dairy heifers for milk production
 Increase reproductive yield of cows that do not respond to
superovulation
 Obtain oocytes from donors in their first trimester (100 days) of
pregnancy
 Recovery of oocytes from pre /post mortem ovaries (slaughterhouse
ovaries)
In-vitro fertilization (IVF) (cont’d)
… IVF Improves reproductive efficiency of genetically elite
female cattle:
 …Normally: Ovulation of one viable oocyte per estrous
cycle (cow)
•… but on the ovary up to 50 antral follicles present at any time
of the estrous cycle

Via OPU:
15-20 oocytes each week
(twice weekly collection of 7-10
oocytes per collection)

 700-1000 oocytes/year/cow
80-120 pregnancies / year /cow
(a reliable IVF system and a dedicated
OPU team are required!!!)
In-vitro fertilization (IVF) (cont’d)

Disadvantages/limitations

 Requires great deal of skill

and details

 Very expensive 1 = Oocyte

2 = Pellucid zone
3 = Stratum granulosum

 Low rates of success 4 = Theca interna

5 = Theca externa
6 = Antral follicle
 Large, abnormal calves with 7 = Cumulus oophorus (Granulosa cells, together with the oocyte)
8 = Basal lamina between theca and stratum granulosum

abnormal placentae born in some

cases –‖ Large Offspring Syndrome‖ (LOS)
In-vitro fertilization (IVF) (Cont’d)

Bovine IVEP in different continent in 2017 (Viana, 2017, 2018)

Steps involved in IVF
1. Oocyte collection and selection for in vitro maturation
– Obtaining of eggs from the ovaries of the female donor
Collection of eggs
under transvaginal
ultrasound guidance
(OPU)

a needle is inserted through the vaginal

wall into the ovaries using ultrasound to
locate each follicle
…the follicular fluid is drawn up into
a test tube to obtain an egg (oocyte) from
the female donor
In-vitro fertilization (IVF) (Cont’d)
Media should simulate the
fluids in the female
reproductive tract

pH

Sterile environment in a
Laminar Air Flow hood Osmolality
...Searching for useful eggs
 Selection is based on the morphology
of the oocyte:
 the degree of COC compaction
 the presence of homogeneous ooplasm
(Sirard et al., 2006) Oocytes with compact multi-
layered cumulus cells (>5)
and homogeneous ooplasm

Oocyte with less compact cumulus

cells and irregular ooplasm
 In-vitro fertilization (IVF) (Cont’d)

2. In vitro maturation of oocytes

 The immature oocytes are incubated in vitro - in maturation
medium: e.g. TCM 199 medium, with 10% fetal calf serum and
gonadotrpins (FSH, LH) … simulate the fluids in the female reproductive
tract
24-48 hours of maturation in incubator

38.5°C
5% CO2
Criteria for matured oocytes
A) Expansion of cumulus cell
- degree of cumulus cell
expansion A

B
B) Extruded first polar body
B

C) Formation of perivitelline
C
space
D
D) Condition of zona pellucida
(thickness, and regularity of its shape)
Source: Asnaku, (MSc thesis 2021)
In-vitro fertilization (IVF) (Cont’d)

1 1-2

3 4
 LH peak Nuclear maturation
Pre ovulatory
follicle

 Dissociation of Cumulus Follicular Rupture /

cells Ovulation

 Meiosis Resumption
ON OFF
OFF
germinal vesicle 1st Polar
GVBD metaphase II
metaphase I Body
2n 4DNAc n 2 DNAc
3. Collection and preparation of sperm

 Sperm preparation / separation - Swim-up or Percoll gradient procedures

 Sperm capacitation – sperm treatment with capacitation - media
•Bovine serum albumin
•Heparin- glucosaminoglycans
•Caffeine – cyclic nucleotide phosphodiesterase ihibitor
 Enhance motility of spermatozoa
 Express the acrosome reaction
 Enhance successful fertilization of the oocytes

4. Insemination of eggs by sperm

 Eggs are taken in small droplets of culture / fertilization medium

 … Insemination- a dose of sperm consists of about one million sperms per

ml of medium …  Co-culture for 6 -24 hours
In vitro Fertilisation

(1 million)

Incubation
+ together for
6-24 hours
In-vitro fertilization (IVF) (Cont’d)

Evaluation for evidence of

fertilization after 24 hours

 If no eggs fertilized, then

insemination of eggs by
intracytoplasmic sperm injection
(ICSI)
ICSI an alternative to
In vitro Fertilisation
(human, horse....)
Embryo incubated for 6 – 7/8 days, before ET
…→will facilitate the formation of morula or blastocyst
60 hours after
30 hours after 40 hours after fertilisation
fertilisation fertilisation

Co- culture (oviductal cells) Synthetic media (Synthetic

Oviductal Fluid) +/- Foetal Calf Serum (growth factors)
 In-vitro fertilization (IVF) (Cont’d)

Fertilisation

Maturation Culture

In vitro 7 days
T0
post fertilisation

-36 hrs (pig) 18 hrs

-24hrs (cow)
IVF and embryo preservation: freezing embryos for
later transfer to surrogate mothers Freezing Transfer to
Freezing of semen and embryos provide recipients
enormous flexibility in using AI and ET techniques
for genetic improvement In vivo
5. Transfer of embryo into uterus
ET - Embryo in the morula/blastocyst stage

Embryos are transferred to the uterus through a fine tube

(catheter), passed through the cervix

Embryos are placed in the top part of the uterus

•2-4 embryos can be transferred at one treatment cycle

 In vitro assisted fertilization-Gamete/Embryo
micromanipulation
…Micro-insemination: bypasses the zona pellucida (in case of
infertility by the conventional in vitro fertilization procedure- human)
1) Sub-zonal insemination (SUZI)  Sperm placed between zp
and vitelline membrane (can lead to polyspermy!)

Diagrammatic representation of sub-zonal injection (SUZI). Several sperm are selected and
injected under the ZP in SUZI (Payne (1995)
2) Intra-cytoplasmic sperm injection (ICSI)
By inserting a needle carrying
a single sperm cell through the
ZP into the oocyte cytoplasma
…bypasses the normal process
of fertilization

successfully applied in various

mammal species Diagrammatic representation of ICSI. The ICSI
involves the injection of a single, live,
immobilized sperm into the ooplasm of the
…technically demanding and oocyte. (Payne (1995)
costly procedure
ICSI an alternative to
In vitro Fertilisation
(human, horse....)
 Methods of Zona pellucida dissection for
micro-insemination:
 Zona drilling with
 an acidified solution (partial
zona digestion)

 an ultraviolet micro beam

(inserting of a sperm through
laser drilled hole with optical
tweezers into the perivitelline
space (PVE)
Diagrammatic representation of Zona Drilling (ZD). Acid Tyrodes solution is
used to make a hole in the ZP to allow sperm access to the oocyte , Catt (1996)
 Partial zona pellucida
dissection with fine
needle

Diagrammatic representation of Partial Zona Dissection (PZD).

A cumulus-free oocyte is held by suction onto a holding pipette and a fine glass
needle is pushed through both sides of the ZP; the oocyte is then released and the
glass needle is pushed against the holding pipette until a slit in the ZP is produced
(Payne (1995)
Sperm sexing
 In mammals, sex is determined by which spermatozoon fertilizes
the ovum: the X- or Y-chromosome–bearing gamete

…Phenotypic sex - predetermined by sorting sperm into X and Y

populations prior to insemination (Johnson, 1992)

Beef industry: male animals – higher feed conversion

efficiency, higher body weight gain
Dairy industry: heifer calves – for production of offspring and
milk
 Sperm sexing (cont’d)

Flow-cytometric measurements differentiate between X and Y

cells, detection of a difference in DNA content of sperm

Separation of X- and Y chromosome-bearing sperm based on

X/Y DNA content deference  sorting at a purity of 95%

E.g. Cattle: X-bearing sperm contain 3.9% more DNA than the Y-
bearing sperm

•The DNA content differences in sperm from bulls, boars, rams,

and rabbits were 3.9%, 3.7%, 4.0%, and 3.9%, respectively
(Garner et al, 1983)
Sperm sexing (cont’d)

Sex-sorted bovine sperm - for in vitro fertilization

(IVF) to generate embryos from in vitro–matured
oocytes (Cran et al, 1993):

 Ovum pick up (ultrasound and guided needle to

aspirate immature oocytes (collection):
•In vitro maturation,
•In vitro fertilization (AI with sexed semen!),
•In vitro cultured in vitro for up to 7 days)
• …suitable for ET or freezing
Sperm sexing (cont’d)
Flow cytometry:- Separation of sperm into X and Y chromosome- bearing
cell fraction relies on the use of fluorescent dye (Hoechst 33342) that is taken
up by the DNA,  the fluorescent dye glows when illuminated with a laser
light

…X-chromosome, with more DNA, gives off more light thus

detected separation using flow cytometry … sorting at a rate of some 10
million/h (each population)
Sperm sexing (cont’d)

 Sexed Semen AI: Insemination of 1–5 x 105 sperm/dose

sorted sperm directly into the uterine horn, deep uterine
insemination … Commonly employed in ET (preferably in
heifers!)
• … bypasses problems of successful traversing of
sperm through the female tract
Success rate – using sexed semen:
25% lower pregnancy rates with sexed, cryopreserved sperm
compared with conventional sperm (DeJarnette et al., 2008)
Reasons:
•lower doses of sperm per straw
•a negative effect of the sorting process
…Sperm sexing produce sexed offspring with 85%–95%
accuracy (Johnson and Seidel, 1999)
Sperm sexing (cont’d)

Cryopreservation of sorted sperm

Cryopreservation of sorted cells: 1x 106 sperm per
dose in 0.25-ml straws (Schenk et al, 1999), …
compensate for cell death due to cryopreservation and
thawing

…The numbers of sperm for routine AI in cattle: 20 X

106 sperm/dose
Embryo sexing
An identical complete set of chromosomes are contained within the
nucleus of:

•a diploid zygote
•every cell of the embryo and the developing fetus
•neonate and mature animal

The essential material of chromosomes is DNA (deoxyribonucleic

acid):
•molecular software that encodes information for the genes on
the chromosomes
•…specifies a particular animal
Embryo sexing (cont’d)

Cells of all individuals of a species contain the same

chromosomes (same genes)
The difference is:
 The cells of a male contain the Y chromosome, that
females lack

 A single gene on the Y chromosome triggers testis

differentiation ―maleness” (SRY, Sex determining
Region of Y -Chromosome)
 Initiates development of male sex organs resulting in
male phenotype
Embryo sexing (cont’d)

…Maleness - a unique (Y) chromosome

Embryo sexing: Polymerise Chain Reaction (PCR)- used

for the assay for Y-chromosomal DNA

An assay for Y-specific DNA — in any cell at any stage in

the life cycle will establish whether that cell is male:
•…amplification of Y- chromosome-specific repetitive
sequences
•  detection of DNA sequences unique to the Y
chromosome identifies a male!
•…the absence of such sequences identifies a female!

 this is the basis of embryo sexing

Embryo sexing (cont’d)

Detection of a single genome

specific to male DNA sequence
from embryo biopsies:

1) Embryo biopsy from in Vivo /

in vitro produced embryos
 Embryo sexing (cont’d )
2) Lysis of cells and DNA extraction

3) Add DNA primer(s) amplification of Y- chromosome-specific

repetitive sequences
4) PCR Amplification of DNA sequence(s)

Male and Autosomal DNA (UNCEIA) Male DNA (AB Tech. Ampli Y)
Amplification of Y- chromosome-specific repetitive sequences
 Cloning
…The use of technology to make an exact genetic
copy of a living organism
Methods:
1) Somatic cell nuclear transfer
2) Nuclear transplantation
3) Embryo splitting
 Cloning (cont’d)

Somatic Cell Nuclear Transfer

Steps:
 Somatic cell from the original organism, donor cells, are grown in the
laboratory (source of genetic material: the body (skin or ear))
 The Egg(s) (ovum) is collected and prepared by removing its genetic material
 The selected donor cell is placed next to the empty ovum and a small
electrical current allows the genetic material from donor cell to fuse into the
ovum

…The ovum, with its complete set of genes, is “tricked” by

thinking it has been fertilized and develops into an embryo
•The embryo is then implanted/ transferred into prepared
uterus
Cloning (cont’d)

Nuclear transplantation

It is a process of making multiple copies of the same

original cell

The blastomeres from one embryo is fused with

prepared egg cells, using an electric current
Nuclear transplantation to make genetically copy animals (Brevini et al., 2008).
Cloning, Embryonic cloning
Enucleate oocytes
Collect Oocytes
IVF, In vitro
development

Fusion
Isolate Reconstituted
blastomeres embryos
Transfer
 Cloning (cont’d)

Embryo splitting
The simplest way to create a clone is splitting of a fertilized
egg into two:

Fertilization … blastomeres
•Using special instruments
zona pellucida is taken away
•The blastomeres are teased
apart and each coated with
an artificial zona pellucida,
start growing as individual embryos …  transferred
recipients
…→a maximum of 4 embryos by using embryo splitting method
Production of transgenic animals - genetic
engineering

Genetic engineering is insertion of a specific piece of foreign

DNA into a cell

Gene splicing is used to introduce

one or more genes of an organism
into a second organism

•…Transgenics, recombinant DNA is the transferal of a

specific gene from one organism to another

•the inserted DNA is able to replicate and pass on to

daughter cells during cell division
Prodn. of transgenic …(cont’d)

Methods for the creation of transgenic animals:

1. DNA microinjection

2. Retrovirus-mediated gene transfer

3. Embryonic stem cell-mediated gene transfer

Prodn. of transgenic …(cont’d)

a) DNA microinjection
direct microinjection of a chosen gene (a single gene or a
combination of genes) from another member of the same
species, into the pronucleus of a fertilized ovum

 The introduced DNA may lead to the over- or under-

expression of certain genes or to the expression of genes
entirely new to the animal species

the manipulated fertilized ovum is transferred into the

uterus of a recipient female
 Applicable to a wide variety of species
Prodn. of transgenic …(cont’d)

b) Retrovirus-mediated gene transfer

 To increase the probability of expression, gene transfer is

mediated by means of a carrier or vector, a virus or a plasmid

 Retroviruses commonly used as vectors to transfer genetic

material into the cell, ability to infect host cells

 →Transmission of the transgene is possible only if the

retrovirus integrates into some of the germ cells

 …→ Offspring derived from this method are chimeric, i.e., not

all cells carry the retrovirus
Production. of transgenic …(cont’d)

C) Embryonic stem cell-mediated gene transfer

 Prior insertion of the desired DNA sequence into an in

vitro culture of embryonic stem (ES) cells

•Stem cells are undifferentiated cells that have the

potential to differentiate into any type of cell, to
give rise to a complete organism
Production. of transgenic … (cont’d)

 Cells are incorporated into an embryo at the

blastocyst stage of development

 The result is a chimeric animal

 ES cell-mediated gene transfer is the method of

choice for gene inactivation, the so-called
knock-out method

 This technique is used for the study of the genetic

control of developmental processes
Production of transgenic animals

What are Stem cells ?

 Self-renew (indefinite multiplication)

 Differentiate into specialized cell types

Symmetrical
division
Production of transgenic animals

Stem cell hierarchy

 Embryonic stem cells (ES
cells)

 Fetal stem cells

 Adult stem cells

Stem cell potency
- the ability to differentiate into other cell
types

?
Cell-based therapy
 Several devastating human diseases are
caused by loss of cells
 Stem cell may be a source of ‖spare‖ cells
for therapy
Applications

 To repear human
(animal ?) tissues....
 Degenerative
Deseases
&
 Injuries
(neurology....,)
Stem cell treatment
Great prospects!!!

Significant risks???
Prospects of iPSC
Cell-based therapy
In vitro cell
Fibroblasts iPSC Neurons model for the
patient

Personalized
treatment

Correction of
Alzheimer’s genetic defect
patient
Stem cell treatment
…the need of an intermediate model!!!
Production of transgenic animals (cont’d)

Application of this biotechnology:

In medical research, to identify the functions of specific

factors in complex homeostatic systems through over- or
under-expression of a modified gene (the inserted transgene)

In toxicology, as responsive test animals (detection of

toxicants)
In mammalian developmental genetics
In molecular biology, the analysis of the regulation of gene
expression makes use of the evaluation of a specific genetic
change at the level of the whole animal
Production of transgenic animals (cont’d)

In the pharmaceutical industry, targeted production of

pharmaceutical proteins, drug production and product efficacy
testing

In biotechnology: as producers of specific proteins

… A transgenic animal is created once the second

organism incorporates the new DNA into its own genetic
material
The inserted DNA is known as transgene and any organism
containing the transgenic is called Transgenic or Genetically
Modified Organism (GMO)
•New proteins are expressed
•Valuable protein in the milk (milk composition)
•Valuable protein in the serum (for therapeutic purpose in humans)… etc.
References
 Reproduction in farm animals (1993). 6th edition. E.S.E. Hafez. Lea &
Febiger, Philadelphia.
 Veterinary reproduction and obstetrics (1989). 6th edition. Geoffrey H.
Arthur; David E. Noakes; Harold Pearson. Bailliere Tindall. London -
Philadelphia - Toronto – Sydney – Toky

Veterinary reproduction and obstetrics (2001). 6th edition. Geoffrey H.

Arthur; David E. Noakes; Harold Pearson. Bailliere Tindall. London -
Philadelphia - Toronto – Sydney – Toky
animal reproduction (2000). 5th edition. H. Joe Bearden; John W. Fuquay.
Prentice-Hall, Inc. Upper Saddle River. USA.