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DEVELOPMENTS AND PROSPECTS OF ADVANCE REPRODUCTIVE

BIOTECHNOLOGIES IN BUFFALO PRODUCTION

Danilda HUFANA-DURAN, Ph.D.

Scientist I, Philippine Carabao Center and

Affiliate Professor, Central Luzon State University

Science City of Muñoz, Nueva Ecija 3120

Philippines

dhduran@pcc.gov.ph

Science City of Muñoz,Nueva Ecija 3120

Philippines

www.pcc.gov.ph

Philippine Carabao Center (PCC)

A premier institution promoting profitable and

sustainable buffalo-based

enterprises designed to improve the income and

nutrition of rural communities

“Towards better nutrition, higher levels of income and improved

general well-being of the rural

farming families… through the

conservation, propagation and promotion of water buffalo as

important source of milk and meat,

in addition to draft power and hide”

Buffalo Inventory, Philippines, 2017

Population: 2.88Million

Reproduction has to be improved and

genetically superior animals be produced.

� Increase human

population

� High extraction rate

Without technical

intervention will

result to huge

problem in the

future.

REPRODUCTIVE BIOTECHNOLOGIES

Male Female

Semen Collection, Processing, and

Cryopreservation

Sperm Sexing

Motile Sperm

Separation

Prediction of

Fertility

Estrus/OvulationSynchronization

MOET/SOET

Ovum pick up

In vitro maturation of

oocytes

Artificial Insemination

In Vitro Fertilization

Intra-cytoplasmic sperm injection

Cloning by Somatic Cell

Nuclear Transfer

Embryo culture/sexing/splittingCryopreservation/transfer

GENETICALLY SUPERIOR OFFSPRING

Pregnancy Diagnosis &

Ultrasonography

� Use of fertility-predicted bulls as donors of semen

� Well-trained village-based AI technicians

� Perfect timing of AI using vaginal cytology (Duran et al., 2015)

Improving ArtificialInsemination Efficiency

Hufana-Duran et al., 2016

Duran et al., 2015

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MULTIPLE OVULATION & EMBRYO TRANSFER

� Hormonal (PMSG or FSH or hCG) administration to increase the follicle availability.

� Ave. ovulations=1.5 to 9

� Ave. embryos recovered= 0 to 4.5

(Drost et al.,1986; Misra et al.,1990;Cruz et al.,1991; Kasiraj et al.,1993(India); Mutha Rao et al., 1994; Kandiet al. 2012 (Egypt); Qin et al. 2012(China) Hufana-Duran and Duran. Bubaline Theriogenology2015.

MULTIPLE OVULATION & EMBRYO TRANSFER

Hufana-Duran and Duran. Bubaline Theriogenology2015.

� Buffaloes has smaller reproductive tract than in cattle

� Poor response to superovulation treatment

� In vivo collection of embryos requires expertise

In Vitro Embryo Production Technology

In Vitro Fertilization

Semen fromsuperior sire

Slaughter House

Cryopreservation

In Vitro CultureIn Vitro Maturation

Grade A& B oocytes

Transport

to

Philippines

StrategyHigh genetics donor (India)

Collect

immature eggs

Mature eggs

in vitro

Fertilize eggs

in vitro

Semen from Superior Sire (India)

High Genetics Embryos

Embryos are

cryopreserved Transfer of embryos to

recipient animals

High Genetics Calves

Strategy

Transfer of embryos to

recipient animals

High Genetics Calves

Riverine Calves from IVP Embryos

�Nature of estrus: Natural, 40%�Stage of embryo : Morula to Blastocyst

�Embryo-recipient synchony

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Riverine Calves from IVP Embryos PRODUCTION AND REPRODUCTION PERFORMANCE OF RESULTANT OFFSPRING FROM IVP-VITRIFIED EMBRYOS

MALE, N=3 FEMALE, N=3

Parameters IVP-

derived

Natural

-derived

Age, 1st

Calving

29-31

mon

29-31

mon

Milk

prod,li/day

8-13 4-15

No. of

calving in 5

years

2-3 2-3

Parameters IVP-

derived

Natural

-derived

Age, semen

donor

3 to 3.5

years

3 to 3.5

years

Vol of

semen/eja

1.8 to 3.6 1.2 to

4.6

Sperm

conc./mL

97 to

158x7

80 to

198x7

Ave. FS

prod/year

2,400 to

4,000

2,000 to

6,000PC: 36-day oldIrfan: IVEP11-day old

Ovum Pick Up

Parameter Range Ave.±SD

Retrieval

rate, %

66 to 100 73.8

No. of

follicle

3 to 13 4.8±1.2

Embryo/

donor/wk.

0.7 to 4.4 1.2

Aquino et al.

Buffalo Bulletin,

2013; 32:545-548

Liang et al., Therio.

2008; 69:822-826

Sperm Sexing

Photo Photo Photo Photo courtesycourtesycourtesycourtesy: : : : SexingSexingSexingSexingTechnologies, USATechnologies, USATechnologies, USATechnologies, USA� sperm sexing provides the opportunity to

produce offspring of pre-determined sex

� creation and development of flow cytometer

� expensive due to the sexing efficiency

� with significant positive impact on dairy and

beef production systems

� calving rates of 51.9% and sex accuracy of

89.0% [Lu K. Buffalo Bull 2013; 32:22-30.

Intra-Cytoplasmnic Sperm Injection (ICSI)

Benefits

� collecting and injecting an

acrosome and membrane-

intact single sperm into the

ooplasm of a metaphase II

oocyte for fertilization

� answer to infertility in bulls

and use of sex-sorted sperm

� alternative procedure to

pronuclear injection for

producing transgenic animals

Challenges

� failure of male pronucleus

formation into generated

embryo (Chankitisakul et al.,

Theriogenology. 2012;

77:1415-1424.

� Highly invasive procedure

requiring expensive

equipment and technical

expertise

Cloning

� production of embryos that are

an exact genetic replica or copy of

the animal desired to reproduce.

� somatic cell nuclear transfer

(SCNT) or embryo splitting

� possibility of reproducing copies

of a super buffalo

� smaller number of animals

needed to produce the required

volume of milk and meat,

benefiting human and the

environment

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Successful Cloning

Enucleation Nuclear transfer

� fetal fibroblasts or granulosa cells as donor cells (Shi et al., 2007),

� ear fibroblast nucleus from river buffalo fused into swamp buffalo

oocyte cytoplasm (Yang et al., 2010)

� hand-made zona-free cloned vitrified embryo derived from

enucleated oocytes reconstructed using adult skin fibroblast cells as

nucleus donor (Shah et al., 2012).

Breakthroughs in advance reproductive biotechnologies in water buffaloes

Biotechniques Calf prod. rate Author

Superovulation 33.3 to 100% Drost et al., 1983; Misra et

al., 1990;Cruz et al., 1991

IVEP – slow freezing 23.1% (9/39) Kasiraj et al., 1993

IVEP-vitrified 10.0-40%

(21/174)

Hufana-Duran et al, 2004;

2005; 2007

Sex-sorted IVF-fresh 200% (2/1) Lu et al., 2007

Cloning-fresh 14.3(3/21) Shi et al., 2007

OPU-Sex sperm 20.6 (7/34) Liang et al., 2008

Embryo Splitting 1.4% (1/73) Zhang et al., 2011

Hand-made cloning 29.2% (7/24) Saha et al., 2013

PROBLEMS/SOLUTIONS

PROBLEMS SOLUTIONS

1. Invasiveness Collaboration &

maximize operation

2. expertise and

skills

Training and practice

3. Cost of

implementation

Improve success rate

4. Buffalo

physiology

Further research

PROSPECTS

LIB

Livestock Improvement Biotechnologists

Ovum Pick-Up

from Genetically Superior Female

Oocyte In Vitro

Maturation in CO2 Incubator

In Vitro Fertilization using

Sperm from Genetically Superior Male

In Vitro Culture for Development of Pre-implantation Stage

Embryos

Cryopreservation of Embryo by Vitrification

Method

Embryo Transfer to

Native Carabao of Farmers

Riverine Buffalo Offspring Born to

Native Carabao

Mobile Laboratory for Field Application

� Propagation and

conservation of

genetically superior

animals

� Improve the success

rate by research and

training

� Establish R&D

collaborations

� Development of

R&D Program on

reproduction

Hufana-Duran D and PG Duran. Advance Reproductive

Technologies in Water Buffalo. In: Bubaline Theriogenology,

Purohit G.N. (Ed.). International Veterinary Information Service,

Ithaca NY (www.ivis.org), Last updated: 30-Sep-2015; A5731.0915

Thank you!!!