psdb 2010 presentation - fin

8/4/2019 PSDB 2010 Presentation - Fin

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DANILDA HUFANA-DURAN, Ph.D.Senior Embryologist

Reproductive Biotechnology LaboratoryPhilippine Carabao Center

Muoz, Nueva [email protected]

THE EXCITING WORLD OF INVITRO PRODUCTION OF

EMBRYOS

ISO 9001:2000


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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 Strengtheningof the Philippine Carabao Research and

Development Center PHI 78/017 and PHI 86/005coordinated by PCARRD became operational in 1993 mandated to conserve,

propagate and promote the carabao as a source ofdraft animal power, meat, milk and hide to benefit

the rural farming families.


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VISION

A premier institutionpromoting profitableand sustainable

carabao-basedenterprises designedto improve the incomeand nutrition of small-scale farmingcommunities


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MISSION

Improve the generalwell-being of ruralfarming communitiesthrough carabao genetic

improvement, technologydevelopment anddissemination, andestablishment of

carabao-basedenterprises thus ensuringhigher income and betternutrition


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PROGRAMS &SERVICES

Genetic improvement

Artificial insemination

Bull loan

Production of high quality

breeding animals

Frozen semen and embryodistribution

Technical assistance

Training Carabao-based

enterprise development

Laboratory services


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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)


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REPRODUCTIVEBIOTECHNOLOGIES


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Production of embryos

Cleavage

Development of pre-implantation stages


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PCC Reproductive Biotechnology


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Equipments


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In VitroEmbryo Production Technology

In Vitro Maturation

In Vitro Culture

CryopreservationEmbryo Transfer

In Vitro

Fertilization

Semen fromsuperior sire

In Vitro Maturation

Slaughter House

ICSISUZI

Cloning

Sexed-

sperm

sexing

Ovum pick-up

or

DDGSS

Geneticallysuperior calf


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Ovum Pick-Up


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In Vitro Fertilization

Thawing of FrozenSemen

Sperm-oocyte co-culture for 6-8 hrs

Retrieval of SpermCells by Centrifugation

Washing of SpermCells

From the incubator,IVM oocytes aretaken out, cumuluscells are going tobe partly removed Mixing of oocytes

with the sperm cells

IVF dish

100 ug Na-Pyruvate10 mM Caffeine4 units heparin

BO Medium

PREPARATION

Discard

supernatant

Sperm pellet

Adjust spermConcentration

(2M/ml)

45/65/95
http://../dhduran/Desktop/0h%20cap%20semn%20COva%206-19.mp4http://../dhduran/Desktop/0h%20cap%20semn%20COva%206-19.mp4


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

Separation of the motilesperm cells from the dead,abnormal population and

seminal contaminants bydiscontinues densitygradient separation (DDGS)

45% CSSP65% CSSP

95% CSSP

Come up with an IVF

environment with purelymotile sperm cells thatcould significantly improve

the IVF success.


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SUZI(Sub Zonal Insemination)

IVF(In Vitro Fertilization)

The technique for assisted fertilization


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Intracytoplasmic sperm injection : ICSI

One sperm is injected into ooplasm

directly by glass pipette. fertilization

malefemale

* We can use immotile sperm for fertilization.

* We can form fertilization with only one sperm.
http://../ICSI.mp4http://../ICSI.mp4


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Sperm Sexing


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Above:

Modified BDcytometer

Left:

Moflo cytometer

Sperm sexingmachineOPU-derivedoocytes are IVFwith sorted spermcells


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+_Enucleation

ofoocytes..\..\p-enu.mpg

RecipientDonor (Super Buffalo)

Collection of ear skin sample

In Vitro Culture of

Clone embryos

Electrofusion: 2 direct pulsesof 200 V/mm for 20 sec, 1

sec apart

Transfer of somatic cellsto enucleated oocytes..\..\p-nt.mpg

Transfer of NT embryos

to surrogate dams

SOMATIC CELL NUCLEAR TRANSFER

Couplets are cultured inmSOF + 10g/ml cyclohe-ximide for 5 h
http://../p-enu.mpghttp://../p-enu.mpghttp://../p-nt.mpghttp://../p-nt.mpghttp://../p-nt.mpghttp://../p-nt.mpghttp://../p-nt.mpghttp://../p-nt.mpghttp://../p-nt.mpghttp://../p-nt.mpghttp://../p-enu.mpghttp://../p-enu.mpghttp://../p-enu.mpghttp://../p-enu.mpghttp://../p-enu.mpghttp://../p-enu.mpghttp://../p-enu.mpg


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ISSUES

High variability of results

Age of the eggs

The growth stage

Size and atresia grade of the

follicleWhich could possibly seen on

morphology of oocytes

The intrinsic quality of the

oocyte is the key factordetermining the oocytesdevelopmental ability to theblastocysts stage


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Oocytes developmental competence

ability of oocyte to produce normaland viable embryo afterfertilization, a condition that

results from both nuclear andcytoplasmic maturation.

it is acquired during

folliculogenesis, the period ofgrowth and maturation viainteractions with somatic cells


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

Loosen CCCC has startedto expand

Compact CC CC are still

compact

In Vitro Maturation Time:

Longer (24-26 h)

Shorter (20-22 h)


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2. Selection based on thegranulation of the ooplasm

Homogeneous/Even the ooplasm is

evenly granulated

Heterogeneous/Uneven theooplasm is notevenly granulated,some part is either

light or dark


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INSTALLING

IMAGE JFOR

IMAGE ANALYSIS

3. Selection based on the size of theooplasm

i.e.


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4. Base on the size of the donorfollicle, mm

1.


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Success Rate of IVEP & ET

Success Indicators

0

10

2030

40

P HN HS F

0

50

100

IVM IVF IVCCalving rate

%

Development rate

IVEP ET


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206 embryos

51.0% (105/205) w/Metaphase

44.7% (92/206) analyzable

47.7% incidence ofchromosome abnormalities

Polyploidy, i.e. triploidy

23.9%Mixoploidy, 11.9%

haploidy, 11.9%

A case of trisomy. Two X

chromosomes (redarrow) and one Y (pinkarrow) are distinct in a 3nplate.


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IVEP MILESTONE

DEVELOPMENT YEAR

Establish the laboratory 1992

Calf out of IVEP of fresh embryo 1996

Develop cryopreservation of embryos by vitrification 1997

Establishment of satellite laboratory in India 2001

Calf out of in vitro produced-vitrified embryo 2002

Calf out of IVEP-vitrified embryos (2n=50) to swamprecipients (2n=48)

2002

Separation of motile sperm cells by discontinuousdensity gradient sperm separation

2003

Twin calves out of IVEP-vitrified embryos 2004

Calf out of OPU-IVEP embryo 2009


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Publications


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Riverine Calves from IVP Embryos


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Propagation of riverine calves both throughriverine and swamp surrogate mothers out of in

vitro produced embryos


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Improve meat and milkproduction


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ACKNOWLEDGEMENTS

To the organizing committee of the 2thPhilippine Society of Development Biology

Symposium for the invitation to deliver thistalk.


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psdb 2010 presentation - fin

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