EMBRYO TRANSFER TECHNOLOGY

 Embryo transfer technology is a process in which

one or more embryos are removed from the
reproductive tract of a donor female and transfered
them to one or more recipient females
 Applicable E.T technology

-1890 Walter Heape

-1950 Jim Rowson (Cambridge, England.)
-1970 – 1980
 In vivo & In Vitro
EMBRYO TRANSFER TECHNOLOGY

 In Vivo
 Selection of Donors
 Superovulation Treatments
 Insemination of Donors
 Flushing of Embryos
 Evaluation of the Quality of Embryos
 Selection of Recepients and Synchronization
 Embryo Transfer
EMBRYO TRANSFER TECHNOLOGY
 Selection of Donors
- Successful Reproductive Performance
- Healthy Reproductive Tracts
- Females with High Genetic Merit
 Superovulation Treatments
- Maximization of the number of follicle and oocyte.
- Protocoles depending on different hormones.
 Insemination of Donors
- Bull with High Genetic Merit
- Sperm with High Quality
 Flushing of Embryos
- Balloon Catheter
- Y junction
- Filter
 Evaluation of Embryos
 Selection of Recepients and Synchronization
 Embryo Transfer
EMBRYO TRANSFER TECHNOLOGY
 In Vitro Fertlizasyon- In Vitro Production
➢ Slaughterhouse Materials

The ovaries taken from the slaughterhouse are kept in ovarian

transport containers with +4 / +37 C temperature, containing PBS or 0.9%
NaCl. Moves to the laboratory for 3 hours
1. Aspiration Method
In the laboratory, aspiration of the taken oocytes by injection into PBS
or TCM199 solutions from ovarian follicles
2. Slicing Method
It is the process of slicing the ovaries brought in the laboratory and
collecting oocytes into petri dishes by washing the ovaries with PBS or TCM199
solutions.
EMBRYO TRANSFER TECHNOLOGY
 In Vitro Fertlization- In Vitro Production
➢ Live Materials

- LOPU (Laparoscopik Ovum Pick-Up)

The process of collecting oocytes from live animal ovaries through the
help of camera systems and equipment.
It is mostly applied in small ruminants and calves.
Hormonal supplements are needed for superovulation.
- OPU (Ovum- Pick Up)
Oocyte collection from live animal ovaries through the USG and
aspiration needle.
Without the need for superovulation, aspiration can be achieved from
donors in relation to the number of waves of follicles.
EMBRYO TRANSFER TECHNOLOGY
IVF Stages
Washing and Rating of Oocytes
A= Oocytes having a homogeneous structure
with more than 5-6 rows of cumulus cells around
B= Oocytes with 2-4 rows of cumulus cells around, or
not having cumulus cells in a small part
C= Oocytes without any cumulus cells around
D= The oocytes surrounded with degenerate cumulus
cells
EMBRYO TRANSFER TECHNOLOGY
 Tubes in which oocytes are collected are emtied into
the petri dish
 Scanning of the cumulus-oocyte complexes (COCs) is
performed under a heated stereo microscope (37 C).
 Oocytes are then put to the oocyte washing solution
by a pipette and washed..
 The maturation of A and B class by

evaluating the collected oocytes taken

into the process.
EMBRYO TRANSFER TECHNOLOGY

 In Vitro Maturation (IVM)

- 20-22 h.
 In Vitro Fertilization

- %50 or higher motility

- 15.000 – 20.000 sperms per an oocyte
- 6- 22 h.
 In Vitro Culture

 Transfer / Freezing
EMBRYO TRANSFER TECHNOLOGY
 İn Vitro Maturation (IVM)
- Oocytes are taken from oocyte washing solution by pipettes. -
- Drops are formed from oil-coated maturation media to prevent
evaporation and contamination.
- Oocytes are released into these drops.
- It is left in the incubator (5% CO2, 38.5 ° C) for 20-24 hours.
Elements Affecting the Maturation;
❖ Physiological status of the oocyte donor
❖ Östrus cycle and follicular wave
❖ Follicle size
❖ COCs
❖ Quality of the oocytes
❖ Maturation time
❖ Temperature, humidity and environmental gas components
❖ Osmotic value of maturation environments
❖ Substances added to the maturation medium
EMBRYO TRANSFER TECHNOLOGY
 Aspiration, Washing ve Maturation Medium Samples
Solution Content Function
Oocyte Buffer Solution Vitamin, aa, purine ve other nutrients
Aspiration
Solution Serume Protein, Preventing sticking

Antibiotic Antibiotic
Buffer Solution Vitamin, aa, purine ve other nutrients

Oocyte Serume Protein, Preventing sticking

Washing
Solutions
Buffer Solution Vitamin, aa, purine ve other nutrients

Maturation Serume Protein, Preventing sticking

Solutions
Hormone Effect on Maturation

Antibiotic Antibiotic

 Common Buffer solution, Serum, Hormone and Antibiotic Samples

Buffer Solution Serum Hormone Antibiotic
TCM- 199 Fotal Calf Serume FSH Penicilin

SOF Bovine Serume LH Gentamisin

PBS Bovine Serume Eustradiol Streptamicin
Albumine
EMBRYO TRANSFER TECHNOLOGY
 Aspirasyon, Yıkama ve Maturasyon Mediums
Solutions Chemicals Function
D- PBS or TCM-199 Vitamin, aa, purine ve other nutrients
+
Oocyte Aspiration FCS (Calf Serume) Protein, Preventing sticking
Solutions +
Antibiotics Antimicrobial
TCM- 199 or SOF Vitamin, aa, purine ve other nutrients
+
Oocyte Washing FCS (Calf Serume) Protein, Preventing sticking
Solutions

TCM-199 (Tissue Culture Vitamin, aa, purine ve other nutrients

M)
+
FCS (Fotal Calf Serume) Protein, Precursor of Hormone
+ Follicle Stimulating Hormone, maturation
Maturation Solutions FSH (Follicle Stimulate H)
+
LH (luteinizing H)
+
Penicilin

Other Mediums
Ham’s F10a
SOF
 EMBRYO TRANSFER TECHNOLOGY
PBS and D- PBS Solutions SOF medium contents

 TCM medium
and added hormones
- - TCM medium contains vitamin,mineral
- aa, purine ve diğer substances.
- - However, it is used by adding FCS,
- FSH, LH, Estrogen and antibiotics.
EMBRYO TRANSFER TECHNOLOGY
 In Vitro Fertilization (IVF)
In Vitro Ferlization is also defined as the stage that includes washing, maturation
and fertilization of oocytes..
Cumulus cells are removed from mature oocytes.
- Pipette
- Hyaluronic Acid
- Vortex

Preparation of Spermatazoons
- After thawing frozen sperm, it is put in sperm washing solution and centrifuged to
separate from the diluent, the cryoprotectant and seminal plasma.
- It is diluted in order to adjust the sperm per oocyte.
- It is kept in the incubator and capacitation is provided..

After the fertilization process, washing is done to remove the sperm that are attached to
the oocyte and to prevent polyspermia.
EMBRYO TRANSFER TECHNOLOGY
In Vitro Fertilization (IVF)
 The overall success of the in vitro fertilization process
requires good control of different steps:
- Selection of the Sperm
- Sperm Capacitation
- Fertilization
 Numerous techniques have been proposed to remove
unwanted semen components and concentrate the motile
sperm fraction in a suspension of known concentration.
- Bio Direct Washing
- Swim- up
- Percoll
 EMBRYO TRANSFER TECHNOLOGY
In Vitro Fertilizasyon (IVF)
o Percoll Gradient
- It is the technique of using motile spermatazoones in fertilization process by
separating from dead cells, somatic cells and non-motile spermatazoons by centrifugation.
- 90% Percoll solution is prepared.
- TALP is mixed with 45% Per 90% Percoll solution in 1: 1 ratio.
- 90% Percoll (2ml) at the bottom, 45% Percoll (2ml) at the middle layer, 0.5 ml
sperm at the top
- Centrifuge at 350 - 450 g for 15-20 minutes

Preparation of the sperm with 10x concentration

 EMBRYO TRANSFER TECHNOLOGY
In Vitro Fertilization (IVF)
 Swim up
✓ The Ca-free Tyrode's Albumin Lactate Pruvant solution (TALP) is used as a
wash solution.
✓ Semen and washing solution are mixed equally
✓ Aliquots of four 0.25 ml dissolved semen are layered under the liquots of the Sp-
TALP medium in plastic tubes..
✓ It is centrifuged in a 5 ml conical centrifuge tube for 10-15 minutes at 1200-1600
rpm (200-300 x g) and discard the supernatant.
✓ It is diluted with Sp-TALP to a volume of 5 ml,
✓ It is centrifuged again at 1200 - 1600 rpm (200-300 x g) for 5-10 minutes and the
supernatant part is removed
✓ It is incubated at 37 ° C for 30 - 60 minutes at an angle of 45 °.
✓ Top-floating motile spermatozoa are collected

NOT: RCF = 1.12 x Radius x (rpm/1000)2

RCF: Relative Centrifugal Force = g –force (g)
Rpm: Revolutions per minute of rotor
EMBRYO TRANSFER TECHNOLOGY
In Vitro Fertilization (IVF)
 Swim up
 EMBRYO TRANSFER TECHNOLOGY
 In vitro Fertilization (IVF) Mediums

As

b- Bracket and Oliphant medium

c- Tyrode’s medium base, albumin, lactate and pyruvate
d- H (high amount of Hepes) + TALP (BGM3)
e- oocyte washing medium before fertilization
f- fertilization medium
 EMBRYO TRANSFER TECHNOLOGY
 İn Vitro Fertilization (IVF) mediums
BO Medium- STOK – A

BO Medium - STOK – B

BO Medium
 EMBRYO TRANSFER TECHNOLOGY
 In Vitro Fertilization (IVF) Mediums
OWS (Oosit Washing Solution)

SDS ( Sperm Dilution Solution)

SWS ( Sperm Washing Solution)

 EMBRYO TRANSFER TECHNOLOGY
 In Vitro Fertilization (IVF)
After maturation, oocytes are transferred to 48 µL
fertilization droplets coated with mineral oil after washing twice in
culture medium.
▪ Oocytes are kept in the incubator for 15 minutes until
sperms are added.

▪ After the sperm are thawed, they are centrifugated at 600 x

g for 5-8 minutes and filtered

▪ After centrifugation, the supernatant part is discarded and

the pellet is re-added in 1 mL of modified Tyrode lactate
and centrifuged again at 300 xg for 2 minutes.

▪ The density is then adjusted to 15000 - 25000 sperm per

drop.

▪ Petri dishes are incubated for 18-22 hours at 38.5 ° C, 5.5%

CO2.
EMBRYO TRANSFER TECHNOLOGY
In Vitro Culture (IVC)
▪ After fertilization, zygotes are placed in drops created with culture solutions
and mineral oils by piping method and placed in incubator with 5% CO 2, 5%
O 2, 90% N 2 medium at maximum humidity.
▪ Drops will be renewed at 72th and 120th hours and blastocyst rates are
checked on the 7th day.
EMBRYO TRANSFER TECHNOLOGY
İn Vitro Culture (IVC)
Solutions Chemicals Functions
TCM-199*** Cr1aa Stok A
NaCl Mineral
SOF*** KCl Mineral
CR1aa (Charles Na Pruvat Enerji
Rosenkrans)*** NaHCO3 PH
Fenol Red pH determination
HECM- 6 Cr1aa Stok B
Embryo Culture
Hemicalsium
Medium CZB Laktat Energy
Cr1aa Medium
Stok A
Stok B
BME
MEM
L- Glutamin Energy
BSA (Acid Free) Antitoxic, Growth, sticking (-)
Antibiotic Contamination
FCS Protein, Binding

 *** mediums that ara used mots widely.

 The chemicals section is written for CR1aa, as other media have been written before.
EMBRYO TRANSFER TECHNOLOGY
 Cryoprotectants
-Protection of the embryo from the adverse effect of crystallization
by providing dehydration before freezing.
- Protection of the cell membrane
- Antitoxic
❖ İntra-cellular Cryoprotectants
Low molecular weight substances
- DMSO Embryotoxic and high osmatic pressure
- Gliserol due to its high salt content
- 1-2-propanediol
- Etilen glikol All cell membrane protection in intracellular structure
- Propilen glikol (PG)
- Polietilen glikol (PEG)
- Etanol
EMBRYO TRANSFER TECHNOLOGY
 Cryoprotectants
❖ Extra-Cellular Cryoprotectants
- Glucose
- Fructose The sugar absorbs the intracellular water
- Fikol using the OB difference without penetrating
- Dextran into the cell, thereby stabilizing and protecting
- Sucrose the cell membrane and cytoplasm.
- Trehaloz
- Laktoz
- BSA Has a high molecular weight compared to sugars
- PVP (polivinil pirrolidon) Reduces the size of the ice crystals.
- PVA (polivinil alkol)
- Other polimers
EMBRYO TRANSFER TECHNOLOGY
 Freezing of Embryos
Controlled Slow Freezing
Embryo is in contact with freezing medium at room temperature, and taking
straws during this period is also included.
- The induction of crystallization in the straw in the freezing device
begins to be induced when it reaches -5 / -9 C with a cooled collet
(13 min).
- Freezing process is carried out by decreasing the device by 0.3-0.6
C per minute from -5 / -7 C to -30 / -35 C, when the crystallization
begins.

- In the straw, sucrose is placed next to the embryo and freezing

medium.

- Gliserol
- DMSO Freezing
- %10 oranında Serum Solution
- %0.4 BSA
EMBRYO TRANSFER TECHNOLOGY
 Freezing of Embryos
❖ Rapid Freezing
• Embryo is dehydrated after normal processes.
• It is put into glucose + sucrose and dehydrated again.
• Embryos are placed in straws and kept in liquid nitrogen vapor for 5
minutes.
• To achieve a successful result with this method, one of the cryoprotectants
that can penetrate into the cell such as glycerol, propanediol, DMSO or
ethylene glycol is between 2 and 4.5 M and one of the cryoprotectants that
cannot penetrate into the cell, such as sucrose, trehalose, lactose or
galactose. Freezing solutions consisting of those mixes should be used.
• Unlike others, extracellular fluid freezes in rapid freezing and an increase
in osmolarity of the freezing solution may occur.

Örnek Vasatlar
EMBRYO TRANSFER TECHNOLOGY
 Freezing of Embryos
 Vitrification
 It is the process of making embryos into a glass-like structure
- An easy technique
- Fast ve low cost
 Vitrification Solution Content

 Etilen Glikol

 Gliserol

 BSA -Çözdürme sırasında

 Merkaptoetonol embryonun hücre
 Linoleik asit albümin membranlarının korunması.
 Sucrose -Çözdürülme işlemi sırasında
toksik etkisinin gitmesi için 0.5
ve 0.25 M sukroz ile
seyreltilemeli !

ha V itrifikasyon sukroz pamuk

va solusonu
EMBRYO TRANSFER TECHNOLOGY
 Thawing of Freezed Embryos
i. Frozen straws are removed from liquid nitrogen and kept in the air for 10
seconds. Then it is expected to thaw by throwing in 30 C warm water.
ii. When the ice crystals become invisible, they are removed from the water
bath and wiped with alcohol.
iii. The straw is cut with scissors and taken into a petri dish with PBS and
serum, which is heated on the heating table.
iv. Embryo is examined and assessed under a microscope and kept in this
solution for 10 minutes.
v. The embryos are then placed in a wash solution containing buffer, serum
and beta-mercaptoethanol to be washed.
vi. Then, it is placed in a new petri dish containing the same solution and
placed in the incubator and checked every 24 hours.
EMBRYO TRANSFER TECHNOLOGY
Advantages

 Getting too many offsprings in a short time

 Animal Breeding

 Scientific Research

 Conservation of Genetic Resources

EMBRYO TRANSFER TECHNOLOGY

Disadvantages!!!!!
 Expensive

 Small number of qualified person

 Low pregnancy rates (between%20-60 depending)

 Donors' failure to respond to hormones

 Variability in the number of embryos obtained