THE EXCITING WORLD OF IN VITRO PRODUCTION OF EMBRYOS

DANILDA HUFANA-DURAN, Ph.D.

ISO 9001:2000

Senior Embryologist Reproductive Biotechnology Laboratory Philippine Carabao Center Muoz, Nueva Ecija danildahd@yahoo.com

THE PHILIPPINE CARABAO CENTER

an attached agency of the Department of Agriculture taking momentum from the gains and achievements of the UNDP/FAO-assisted projects Strengthening of the Philippine Carabao Research and Development Center PHI 78/017 and PHI 86/005 coordinated by PCARRD became operational in 1993 mandated to conserve, propagate and promote the carabao as a source of draft animal power, meat, milk and hide to benefit the rural farming families.

VISION
A premier institution promoting profitable and sustainable carabao-based enterprises designed to improve the income and nutrition of smallscale farming communities

MISSION
Improve the general well-being of rural farming communities through carabao genetic improvement, technology development and dissemination, and establishment of carabao-based enterprises thus ensuring higher income and better nutrition

PROGRAMS & SERVICES

Genetic improvement

Artificial insemination Bull loan Production of high quality breeding animals Frozen semen and embryo distribution

Technical assistance Training Carabao-based enterprise development Laboratory services

GENETIC IMPROVEMENT
Gene Pool of Murrah (Dairy) at
- PCC Headquarters, Munoz, Nueva Ecija - PCC at CMU, Bukidnon - PCC at MLPC, Zamboanga

Gene pool of AmBuff (Meat) at

- PCC at USF, Bohol

Gene Pool for Native Carabao

-PCC at CSU, Isabela

Establish gene bank (semen, embryos, somatic cells of genetically superior buffaloes)

REPRODUCTIVE BIOTECHNOLOGIES

Production of embryos
Cleavage

Development of pre-implantation stages

PCC Reproductive Biotechnology

Equipments

In Vitro Embryo Production Technology

or
Ovum pick-up

Sexedsperm Semen from superior sire

ICSI SUZI

DDGSS

Slaughter House

In Vitro Fertilization Cloning

In Vitro Maturation

sexing In Vitro Culture

Genetically superior calf

Embryo Transfer

Cryopreservation

Ovum Pick-Up

In Vitro Fertilization

IVF dish

100 ug Na-Pyruvate 10 mM Caffeine 4 units heparin BO Medium

Thawing of Frozen Semen

45/65/95

Washing of Sperm Cells

Retrieval of Sperm Cells by Centrifugation

PREPARATION

From the incubator, IVM oocytes are taken out, cumulus cells are going to be partly removed

Mixing of oocytes with the sperm cells

Adjust sperm Concentration (2M/ml)

Discard supernatant Sperm pellet

Sperm-oocyte co-culture for 6-8 hrs

Discontinues Density Gradient Sperm Separation (Hufana-Duran et al., 2005)

Separation of the motile sperm cells from the dead, abnormal population and seminal contaminants by discontinues density gradient separation (DDGS) Come up with an IVF environment with purely motile sperm cells that could significantly improve the IVF success.

45% CSSP 65% CSSP 95% CSSP

The technique for assisted fertilization

IVF
(In Vitro Fertilization)

SUZI
(Sub Zonal Insemination)

Intracytoplasmic sperm injection : ICSI

female

male

One sperm is injected into ooplasm directly by glass pipette.

fertilization

* We can use immotile sperm for fertilization. * We can form fertilization with only one sperm.

Sperm Sexing

Sperm sexing machine

OPU-derived oocytes are IVF with sorted sperm cells

Above:

Modified BD cytometer
Left: Moflo cytometer

SOMATIC CELL NUCLEAR TRANSFER

Donor (Super Buffalo)

Recipient

Collection of ear skin sample

Enucleation of oocytes..\..\ p-enu.mpg

Transfer of somatic cells to enucleated oocytes..\..\p-nt.mpg

+ _

Electrofusion: 2 direct pulses of 200 V/mm for 20 sec, 1 sec apart

Couplets are cultured in mSOF + 10g/ml cycloheximide for 5 h

Transfer of NT embryos to surrogate dams

In Vitro Culture of Clone embryos

ISSUES

High variability of results

Age of the eggs The growth stage Size and atresia grade of the follicle Which could possibly seen on morphology of oocytes

The intrinsic quality of the oocyte is the key factor determining the oocytes developmental ability to the blastocysts stage

Oocytes developmental competence

ability

of oocyte to produce normal and viable embryo after fertilization, a condition that results from both nuclear and cytoplasmic maturation.

it is acquired during folliculogenesis, the period of growth and maturation via interactions with somatic cells

1. Selection base on the compactness of the surrounding cumulus cells (CC)

In Vitro Maturation Time:

Compact CC CC are still compact

Longer (24-26 h)

Loosen CC CC has started to expand

Shorter (20-22 h)

2. Selection based on the granulation of the ooplasm

Homogeneous/Even the ooplasm is evenly granulated
Heterogeneous/Un even the ooplasm is not evenly granulated, some part is either light or dark

3. Selection based on the size of the ooplasm i.e. <100, 100-119, 120-139, 140 m
INSTALLING

IMAGE J
FOR IMAGE ANALYSIS

4. Base on the size of the donor follicle, mm

1. 2. 3. 4. 5.

<2-, 2 to 3.94 to 5.96 to 7.98- mm

Success Rate of IVEP & ET

IVEP
100
40 30 20 10 0

ET

% 50
0 IVM IVF IVC

HN

HS

Development rate

Calving rate

Success Indicators

206 embryos 51.0% (105/205) w/ Metaphase 44.7% (92/206) analyzable 47.7% incidence of chromosome abnormalities Polyploidy, i.e. triploidy A case of trisomy. Two X 23.9% chromosomes (red arrow) and one Y (pink Mixoploidy, 11.9% arrow) are distinct in a 3n plate. haploidy, 11.9%

IVEP MILESTONE
DEVELOPMENT
Establish the laboratory Calf out of IVEP of fresh embryo Develop cryopreservation of embryos by vitrification Establishment of satellite laboratory in India Calf out of in vitro produced-vitrified embryo Calf out of IVEP-vitrified embryos (2n=50) to swamp recipients (2n=48) Separation of motile sperm cells by discontinuous density gradient sperm separation Twin calves out of IVEP-vitrified embryos

YEAR
1992 1996 1997 2001 2002 2002 2003 2004

Calf out of OPU-IVEP embryo

2009

Publications

Riverine Calves from IVP Embryos

Propagation of riverine calves both through riverine and swamp surrogate mothers out of in vitro produced embryos

Improve meat and milk production

ACKNOWLEDGEMENTS
To the organizing committee of the 2th Philippine Society of Development Biology Symposium for the invitation to deliver this talk.