state of the art of reproductive biotechnologies in buffaloes4r... · estrus 2-5 days induce...

State of the Art of Reproductive

Biotechnologies in Buffaloes

Embryo Technology and Stem Cell Research Center

Suranaree University of Technology, Thailand

Rangsun PARNPAI, Ashit Kumar PAUL, Yuanyuan LIANG,

Kanchana PANYAWAI, Kanokwan SRIRATTANA, Nucharin SRIPUNYA,

Apichart NGERNSOUNGNERN, Piyada NGERNSOUNGNERN

and Mariena KETUDAT-CAIRNS

3

Contents

2. Estrus/ovulation synchronization

3. Sperm sexing

4. Multiovulation and embryo transfer (MOET)

6. In vitro embryo production (IVEP)

7. Intracytoplasmic sperm injection (ICSI)

5. Embryos sexing

8. Cryopreservation of oocytes and embryos

9. Somatic cell nuclear transfer (SCNT) or Cloning

10. Overall summary

1. Introduction

Genomic technology

Transgenesis

Stem cells

NOT cover

4

Draught power

Buffalo is our daily life

Milk production

FertlizerSocial activities

Transportation

Leather factory

Dung as fuelMeat production

1. Introduction

5

1. Introduction

Buffalo reproductive biotechnologies

Produce more calves

Improve genetics

Reduce calving interval

More female calves for milking buffaloes

More male calves for fattening

Select elite male and female: Conventional vs Genomic screening

6

Year Events Ref.

1983 World’s first ET calf born Drost et al.

1989 Cryopreservation of embryos Techakumphu et al.,

1991 First IVF calf born Madan et al.,

1993 Birth of calf from frozen-thawed embryos (in vivo embryos) Kasiraj et al.,

1993 Sexing embryos (blastomere) by PCR Rao et al.,

1998 Production of OPU-IVF derived embryos Galli et al.,

1999 Production of cloned embryos Parnpai et al.,

2003 Pregnancies of IVF-derived + vitrified embryos Neglis et al.,

2004 Birth of calf from IVF-derived + vitrified embryos Hufana-Duran et al.,

2005 Birth of calf from AI with sexed sperm Presicce et al.,

2005 Vitrified cloned embryos Laowtammathron et al.,

2006 Sexing embryos by Loop-mediated isothermal PCR Hirayama et al.,

2007 World’s first live cloned calf birth Shi et al.

2007 Birth of twin female calve from IVF + sexed sperm Lu et al.,

2008 Birth of calf from OPU-IVF with sexed sperm embryos Liang et al.,

Milestone of reproductive biotechnologies in buffalos 1. Introduction

7


1. Low efficiency of estrus detection, silent heat

2. Seasonality

3. High incidence of postpartum anestrous

Low AI rate

Limited use of artificial insemination

8

Primitive estrus synchronization

Inject PGF2α once or twice Random ovulation and need heat detection

Bachlaus et al., 1980 (India): PR = 27% But Shows estrus 60%

Estrus 2-5 days

AI at estrusInduce luteolysis

Inject PGF2α

if CL presence

Ultrasound check

follicle development

GnRH + AI if DF present Induce

luteolysis

1st PGF2α

after d 5 of the

estrus cycle

De Rensis and Lopez-Gatias, 2007 (Italy): PR = 48 %

13 days 2 days

Induce

luteolysis

2nd PGF2α


9


Ovulation synchronization for fixed time insemination

AI without estrus detection

Save time and high efficiency

P4

Follicular dynamics: Ovsynch

2

6

10

14

Day 9

PGF2a

Day 7

mm

Day 0 Day 1/2

GnRH GnRH FTAI

Day 10

Ov: 30h

16h


0

2

4

6

8

10

20 26 32 38 44Ov ulation (hours)

No o

f buffa

lo

Distribution of the ovulation after the second GnRH

injection (mean of 32.0 5.7h) in buffalo treated with the

synchronization of ovulation protocol (Ovsynch; n=26). Baruselli et al., 1999

88.2% (15/17)

ovulated at 26-38 h

after 2nd GnRH

12h


50.9 48.247.2

0

10

20

30

40

50

60

< 60 days 60 to 99 days > 100 days

(170/334) (158/328) (144/305)

%

Postpartum period and pregnancy rates after synchronization of ovulation

for fixed time artificial insemination in buffalo (n=967)

Baruselli et al., 1999

12 month inter

calving interval


48.8

6.9

0

10

20

30

40

50

60

Breed season Off breeding season

(472/967) (06/86)

%


a

b

Breeding season and pregnancy rates after synchronization of ovulation

for fixed time artificial insemination in buffalo.


How to improve?

Synchronization of ovulation

with progesterone (P4)

and estradiol (E2)


P4 intravaginal device


CIDR-B®

2

8

10

12

Day 11

PGF2a

Day 9

mm

Day 0 Day 3 to 5

EBGnRH,hCG or LH

Synchronized

ovulation

P4

FTAI


Selection of animals

Body condition score

Free from disease

Minimum stress during treatment and AI

Breeding season

Skill and experience of AI technician

Psychological acceptance of animal

17

Factors of success in AI


18

Conclusions estrus/ovulation synchronization


Ovsynch + FTAI is effective to get high pregnancy rate

which became popular in buffalo production industry.

19

3. Sperm sexing

Milking buffalo: Female calves

Beef buffalo: Male calves

XY

Human

X-Y=2.8%

XY

Rabbit

X-Y=3.0%

XY

Boar

X-Y=3.6%

XY

Bull

X-Y=3.8%

XY

Dog

X-Y=3.9%

XY

Stallion

X-Y=3.7%

Fre

qu

en

cy

DNA content

Different of DNA in male and female sperm

Difference in DNA content between X and Y sperm in buffalo

was 3.6% with a sex accuracy of about 90%.

3. Sperm sexing

How can flow cytometry separate X from Y sperm?

DNA as a marker

3. Sperm sexing

- +

Difference charges at sperm cell surface

3. Sperm sexing

sperm orientation:

- One by one

- Flat stream

- Not too fast

3. Sperm sexingFlocytometer sorting sperm:

24

AI with sexed or non sexed sperm

in buffalo

3. Sperm sexing

25

Year Type of buffalo Estrus

status

Pregnancy Calving

(%)

Ref.

Sexed

(%)

Non sexed

(%)

2005 Mediterranean Synch 13/30

(43.3)

9/21

(42.8)NA Presicce et al., 2005


(38.83)

40/106

(37.74)NA Campanile et al.,

2011

2013 Murrah/Nili-Ravi Natural NA NA 3,863/2,006

(51.9)

89% Female

Lu et al., 2013


(49.3)

33/73

(45.2)NA Campanile et al.,

2013


(31.2)

78/142

(54.9)

NA Gaviraghi et al.,

2013

Progress of buffalo AI using sexed or non sexed sperm

3. Sperm sexing

26

Presicce et al., 2005

Buffalo type: Mediterranean (heifers); N= 51

Dose of semen: 2.5 million/dose (Sex sorted sperm)

20 million/dose (Non sexed sperm)

AI: Near the utero tubal jungtion (UJT)

Pregnancy (ultrasound D60-67): Sexed-UJT 13/30 (43.3%)

Non sexed-body 9/21 (42.8%)

Low doses of sex sorted sperm deposited near the UTJ

giving conception rates similar to those of conventional

AI with full dose.

3. Sperm sexing

The first report of AI with sex sorted sperm

27

1,786 were females with 89% of sex accuracy.

Large scale field trial using sexed sperm AI in China

Period: Two years during April, 2009 to March 2011.

A total of 2,006 calves (51.9%, 3,863/2,006) were born.

Lu et al., 2013. 10WBC

Deposited semen at deep uterine horn ipsilateral to

large follicle site.

Semen from Nili-Ravi and Murrah bull.

Swamp buffalo females (heifer and pluriparous)

belong to small farm scale and natural heat.

3. Sperm sexing

28

Buffalo type: Mediterranean (heifers); March – May

Dose of semen: 2 million/dose (Sexed sperm) VS

20 million/dose (Non sexed sperm)

Pregnancy (ultrasound D45 ): Sexed-horn 34/105 (32.38%)

Sexed-body 46/101 (45.54%)

Non sexed-body 40/106 (37.74%).

Campanile et al., 2011

379 heifers

Sexed sperm: horn (105)

Non sexed: body(106)

Sexed sperm: body (101)

3. Sperm sexing

AI with sexed vs non sexed sperm in buffalo heifers

29

3. Sperm sexing

Conclusions sperm sexing

1. Pregnancy rates after AI, not differ between sexed and non

sexed sperm. “Calving rates from AI with sexed sperm were

lower than non sexed sperm”

2. AI in uterine body or deep in uterine horn is effective for sexed

sperm and can be used in lower dose (2 106/dose) compare with

non sexedsperm (20-25 106/dose).

3. AI with sexed sperm, heifers is more pregnancy rates than

cows.

4. Multiovulation and embryo transfer

(MO-ET)

30

First success of MO-ET

Drost et al., 1983 Drost et al., 1988 Parnpai et al., 1985

Chantaraprateep et al., 1989

USA Bulgaria Thailand

Italy

Zicarelli et al., 1992Mishra et al., 1990

India Brazil


31

4. MO-ET

Embryo transfer in buffalo(Univerity of Sao Paulo results)

No. of superovulated No. of embryo

buffaloes recovered

92 1.7/head

Low efficiency: can not commercialize

Baruselli et al, 1994First buffalo born by ET in Latim America (1994)

4. MO-ET

33

4. MO-ET

Does buffalo respond to superovulatory treatments?

R-ovary L-ovary

Baruselli et al, 1994

Injected FSH

34

Post mortem evaluation of ovaries and flushing

of uterus and oviduct in superovulated buffalo

Are the embryos into the uterus?

4. MO-ET


35

R-Ovary L-Ovary

No. CLs after superovulation

4. MO-ET


9.2

3.2

0

2

4

6

8

10

Number of ovulations Number of receverd

oocyte and embryos

Superovulatory responses in buffalo after slaughter and flushing

of oviduct and uterus on day 5.5 (n=12).

35.4%


4. MO-ET

v

Buffalo Bovine

Hypothesis: Some of buffalo oocytes did not

get into fimbria after ovulate

4. MO-ET


38

ET efficiency between buffalo and cattle

5.8 transferable embryo

per flushing

Conception rate

(50%)

2.9 pregnancies

per flushing


per flushing

Conception rate

(30%)


per flushing


4. MO-ET

39

4. MO-ET

Conclusions MO-ET

MO-ET still has problem on very low embryos recovery rate

and can not do commercial application.

Need to do more study in order to improve embryo recovery.

40

1. H-Y antigen (White et al., 1987; Ramalho et al., 2004).

2. Karyotyping (Seike et al., 1990; King et al., 1991; Kittiyanant et al., 2000).

3. Fluorescence in situ hybridization (FISH) (Lee et al., 2004).

4. Polymerase chain reaction (PCR) (Herr et al., 1990; Thibier and Nibart, 1995).

5. Loop mediated Isothermal amplification (LAMP)(Hirayama et al., 2004)

Bovine

Buffalo

1. PCR (Appa Rao et al., 1993).

2. LAMP (Hirayama et al., 2006)

5. Embryos sexing

41

1. Preparation of Samples

1.1 Embryos biopsy

1.2 DNA extraction

3. Subjected to PCR machine

Amplification 2-3 h

4. Analysis of PCR products

Procedures of PCR method

2. Add PCR reaction mixture

F M F M F M F M F M M F F M M

5. Embryos sexing

42

Sexing buffalo embryos using PCR method

5. Embryos sexing

43

0

20

40

60

80

100

120

Morula to blastocyst 16C 4 to 8C

The efficiency of DNA amplification from Murrah buffalo embryos sexing

with PCR method in different developmental stages (4 to 8 cell, 16 cell and

morula to blastocyst) (Appa Rao et al., 1993).

Whole embryo sexingE

ffic

ien

cy o

f D

NA

am

pli

fica

tion

(%

)

100%

(n=30) 85%

(n=20)

55%

(n=18)

5. Embryos sexing

44

Progress of embryos sexing in buffalo by PCR method

Year Method Efficiency of DNA

amplification

(%)

Stage of

embryos

Biopsy method/

No. of blastomeres

Primers/ Type

of buffalo

ET Ref.

1993 PCR 4-8C(55.5),

16C(85)

Mor to Blast (100),

4-8C, 16C,

Mor to

Blast

Whole embryo BRY.I, BRY4.a,

BOV97M/

Murrah

NA Appa Rao et al.,

1993

1998 PCR 2-4C(100)

8-16C(100),

Mor (100),

Blast(100)

2-4C, 8-

16C, Mor ,

Blast

Whole embryo BRY.I, BuRY.I/

NA

NA Appa Rao and

Totey, 1993

2003 PCR 2C(96), 4C(100),

8C(100), 16C(100),

Mor(97),

Blast(94.7)

2C, 4C, 8C,

16C, Mor,

Blast

Whole embryo BRY4.a, BRY. I/

NA

NA Manna et al.,

2003

2007 Multiplex-

nested

PCR

2 to 3C(100) 16 to 32C,

Mor,

CM/

Glass needle under

micromanipulator/

2-3C

SRY/Swamp

buffalo

NA Fu et al., 2007

IVF-derived embryo

5. Embryos sexing

45

1. Preparation of Samples

1.1 Embryos biopsy

1.2 DNA extraction

3. Subjected to heat block or water bath

4. Analysis of PCR products

Procedure of LAMP method

2. Add PCR reaction mixture

+ -

5. Embryos sexing

Amplification 60 min

at 60-65o C

46

A B

Both of the reactions were positive,

the sex of the embryos was judged

as male (A)

Only the common reaction was positive,

the sex of the embryos was judged as

female (B).

Hirayama et al. (2004)

Male positive control Male positive control

5. Embryos sexing

47

Sexing buffalo embryos using LAMP method

5. Embryos sexing

48

No. of blastomeres

100%

Effect of cell number on sensitivity and accuracy of LAMP-based water buffalo

embryo sexing (n= 25 per groups) (Hirayama et al., 2006).

(N=17)

Number of correctly determined (confirmed by the PCR)

82

84

86

88

90

92

94

96

98

100

102

1 2 3 4 5

88%

(n=25)

96%

(n=25)

96%

(n=25)

100%

(n=25)

100%

(n=25)

49

Year Method Source

of

embryo

Efficiency

of DNA

amplificati

on (%)

Accuracy

(%)

(comparing

with PCR)

Stage of

embryos

Biopsy method/

No. of

blastomeres

Primers/

Type of

buffalo

ET Ref.

2006 LAMP SCNT 1C(100),

2C(100),

3C(100),

4C(100),

5C(100)

1C(88),

2C(96),

3C(96),

4C(100),

5C(100)

8 to 16C Glass needle

under

micromanipulator

/1 to 5C

SBuRY.2/

Water buffalo

NA Hirayama et

al., 2006

2011 LAMP

(improve

the

detection

system)

IVF

derived

embryo

1C(100),

2C(100),

3C(100),

4C(100),

5C(100)

1C(70),

2C(80),

3C(80),

4C(100),

5C(100)

Mor Microblade under

micromanipulator

/1 to 5C

SBuRY.2/

Water buffalo

NA Zoheir and

Allam, 2011

Progress of embryos sexing in buffalo by LAMP method

5. Embryos sexing

50

Conclusions embryo sexing

1. LAMP or PCR are equal accuracy and high sensitivity for

embryos sexing.

2. PCR is required the operation time for 2 to 3 h vs. LAMP

only 1 h and PCR needed expensive machine.

5. Embryos sexing

51

6. In vitro embryo production (IVP)

Technique Ref.

IVF (First calve born) Madan et al. 1991

OPU+ IVF embryos production Galli et al. 1998

IVF+ vitrified embryo (Pregnancies) Neglia et al. 2003

IVF + vitrified embryo (calves born) Hufana-Duran et al. 2004

IVF + Sexed sperm (calves born) Lu et al. 2007

OPU+ sexed sperm + IVF (calves born) Liang et al. 2008

Milestone of in vitro embryo production in buffalo

Live

animal

Slaughtered

animal

Ovum pick-up Aspiration

Source of oocytes

6. IVP

Important consideration:

Presence of cumulus cells (Gasparrini et al., 2007)

Follicular fluid (Nandi et al., 2004)

Supplement: Cysteamine (Gasparrini et al., 2003)

βME (Hammam et al., 2010) cysteine (Gasparrini et al., 2003)

Duration of IVM: 16-24h (Gasparrini et al., 2003)

In vitro maturation

6. IVP

54

Sperm viability and capability (Muer et al., 1988; Totey et al., 1992)

Time of insemination (Ravindranath et al., 2003)

Duration of co-incubation (Gasparrini et al., 2007)

Presence of cumulus cells (Attanasio et al. 2010)

Components of Capacitation media: BSA, heparin, caffeine, penicillamine, hypotaurine, calcium inophore (Gasparrini et al., 2006)

IVM medium, oocytes quality, IVC system

Factors affecting success of IVF

6. IVP

Oviductal cells

Buffalo rat liver cells

Cumulus cells

Granulosa cells

In vitro embryo culture

Complex medium Defined medium

SOF, TCM199

co-cultured with

somatic cell

KSOM

CR1aa

mSOFaa

6. IVP

56

Buffalo

type

Source

of

oocytes

Sexed/Unsexed

sperm

BL

(%)

Preg. Ref.

Murrah/Nili-Ravi SH sexed 20 2 Lu et al. 2007

unsexed 28 NA

Murrah/Nili-Ravi OPU sexed 15 9 Liang et al. 2008

unsexed 19 7

Murrah/Nili-Ravi SH sexed 16 NA Liang et al. 2008

OPU sexed 24 NA

Mediterranean OPU unsexed 16.7 NA Boni et al. 1996

Mediterranean OPU unsexed 29.7 NA Neglia et al. 2003

Mediterranean SH unsexed 27 NA Attanasio et al.

2010

Mediterranean OPU unsexed 38.5 51 Saliba et al.2011

IVP in buffalo from sexed/unsexed sperm 6. IVP

57

Slaughterhouse derived oocytes,

IVF with sex sorted semen

Twin female calves birth

on February 13, 2006

World’s first record

6. IVP

Conclusions IVP

The current limitation on buffalo IVP is the low

number of oocytes recovered.

The optimization of the oocytes cryopreservation

efficiency may lead to increased oocytes availability

for IVP.

The use of sex sorted sperm for IVP may further

optimize the competitiveness of this technology.

6. IVP

59

OPU + IVP

Transvaginal ultrasound-guided

for ovum pickup (OPU)

61

Sperm/embryo No. embryos

ET

No. pregnancies

(%)

No. live birth

Male Female

Sexed/fresh 34 9 (26.5) 1 6

Sexed/frozen 43 5 (11.6) 0 4

Unsexed/fresh 26 7 (26.9) 2 3

Unsexed/frozen 39 6 (15.4) 2 3

World’s first calves born

from OPU-IVP

using sexed sperm

6. IVP

42

37

15

5.1

22

15

5

1

18

14

40.6

0

5

10

15

20

25

30

35

40

45

Nellore

(Bos indicus)

Holstein

(Bos taurus)

Buffalo

(Bubalus bubalis)

Visualized follicles

Oocyte recevered

Cleaved embryos

Blastocysts yied

Effect of genetical group (Buffalo, Holstein and Nellore)

on OPU and IVF production. Gimenes et al., 2008

a b b

Buffaloe calf born by OPU-IVP (2011)


6. IVP

Number of OPU 199

Number of oocyte 984 (4.9/OPU)

Number of viable oocyte 584 (2.9/OPU)

Blastocyst rate 38.5%

Conception rate (fresh embryos) 43% (51/119)

Pregnancy loss (30 to 60 days) 4%

OPU-IVP efficiency

Saliba et al., 2011

6. IVP

Conception rate (vitrified embryos) 37.1% (26/70)

Pregnancy loss (30 to 60 days) 5.7% (4/70)

Pregnancy loss (60 to calving) 7.5% (5/66)

Total pregnancy loss 12.8% (9/70)

Saliba et al., 2013

OPU-IVP efficiency

6. IVP

66

OPU-IVP efficiency in buffalo

Blastocyste rate

(30%)

Conception rate

(30%)

9 viable oocytes recovered

2.7 embryos

0.8 pregnancies/OPU-IVP

6. IVP

1. Low number of follicles recruited per follicular

wave (Baruselli et al. 1997)

2. Poor oocytes quality (only 27.3%-31.3% of

recovered oocytes are classified as viable) (Campanile et al. 2003)

3. More fragile zona pellucida (Mondadori et al.

2010), more fragile bonding between cumulus

cells and oocytes (Ohashi et al. 1998)

67

Problems related to OPU+IVP

6. IVP

68

Collection rate by hormone treatment

Treatment Collection rate

by H. treatment

Collection rate

from control

Ref.

FSH 82% 52% Techakumphu et al.

2004

FSH 69.5% 53.1% Promdireg et al.

2005

Hormone treatment gave higher numbers and

good quality oocytes

6. IVP

69

% B

last

ocy

sts

10

5

FSH treated

Non-FSH treated

Oocytes collected

25

20

15

10

5

OPU-IVP efficiency of FSH and non-FSH treated donors

Blastocysts

Parnpai et al., 2014

6. IVP

70

Donor screening base on antral follicles in ovaries

Good Not good

6. IVP

Conclusions OPU-IVP

OPU combined with IVP is a promising technology

to produce the embryo from selected donor.

This technique has the potential to enhance genetic

progression through the female lineage in buffaloes.

Screening donors which have high number of antral

follicles to get high response of FSH treatment and

led to high oocytes recovery rate.

6. IVP

72


• Slow freezing

- Conventional cryoprotectant

Slow cooling of cells to prevent intracellular ice formation

18-35% maturation rate, 4.5% Morula rate (Gautam et al. 2008)

• Vitrification

- A process that produces a glasslike solidification of living cells

that completely avoids ice crystal formation during cooling

73

1M CPA 6M CPA

LN2

-196 C

1. Slow freezing 2. Vitrification

Isotonicsolution

Freezing solution

Coolingslowly

-40 C

Ice formation

Immersedirectly in to LN2

- Cryoloop (Teng et al. 1990)

- Electron microscope grid (Martino et al. 1996)

- Open pulled straw (OPS) (Vajta et al. 1997)

- Solid surface vitrification (SSV) (Dinnyes et al. 2000)

-Microdrop vitrification (Papis et al. 2000)

- Cryotop (Kuwayama et al. 2000 )

- etc. 74

Vitrification devices


7575

Slow freezing vs. Vitrification

Morulae rate 4.3% 11.5%

Blastocyst rate 0.6% 5.4%

Vitrification works better than slow freezing method !


76

GV

- membrane cover the chromosome (condensed)

- microtube, mitochondria, ER were injured after

warming (which is importance for the IVM)

- cumulus cell were denuded

MII

- spindle was sensitive to the change of

tempemrature, easy to deassemble during

cooling lead to chromosome abnormal

76

Source of oocytes freezing7. Cryopreservation of oocytes and embryos

77

Cleavage and blastocyst rate after IVF from freezing GV

stage oocytes after warming is lower than that of MII.(Sharma and Loganathasamy 2007)

EG+DMSO gave the highest maturation rate of buffalo GV

oocytes (Mahmoud et al., 2010)

CB did not improve maturation rate of vitrified GV buffalo

oocytes (Liang et al., 2012)

CPA mixtures may have some advantages over solutions

containing only one permeable CPA (Sharma et al., 2007)

Penetrability of GV membrane (water, CPAs) is lower than MII


Buffalo embryo vitrification at different stages

78

Embryo stages No. (%) embryos

hatched

Morula 10 (91)

Early blastocyst 16 (80)

Blastocyst 15 (75)

Expanded blastocyst 18 (90)

Hufana-Duran et al., 2004

No different was observed on hatching rate in vitrified-warmed

embryos of different developmental stages.


Live birth of in vitro-derived vitrified-warmed buffalo

embryos after embryo transfer

No. of

embryos

transferred

No. of

recipient

animals

No. (%)

pregnant

No. (%)

live births

1 17 1 1(5.8)

2 36 7 4(11.1)

3 2 1 1(50)

Hufana-Duran et al., 2004


Conclusions cryopreservation of oocytes and embryos

1. Vitrification works efficiently than slow freezing

2. Vitrification of MII buffalo oocytes worked better than GV

3. Oocytes/embryo quality affect the cryopreservation efficiency


81

8. Intracytoplasmic sperm injection (ICSI)

Live offspring obtain from ICSI

Animal Activation Ref.

Cattle Yes Goto et al. 1990

Human No Palermo et al. 1992

Mouse No Kimura and Yanagimachi, 1995

Sheep Yes Catt et al. 1996

Horse Yes Cochran et al. 1998

Cat No Pope et al. 1998

Monkey No Hewitson et al. 1999

Pig Yes Martin 2000

Buffalo Yes Liang et al. 2011

8. ICSI

Calcium oscillation during natural fertilization

10 min after sperm-oocytes fusion

8. ICSI

Problems of ICSI in buffalo

Dark cytoplasm

High injection failure

Difficult to activate oocytes

High PA embryo rate

8. ICSI

85

Chemical Activation Treatments Blastocyst rate6-DMAP 29%

EtOH + CHX 24%

The first report of buffalo ICSI:

Liang et al. 2011

8. ICSI

86

Buffalo MII oocytes vitrified by the Microdrop

method yielded 11% blastocyst rates after ICSI

compared with control (23%).

8. ICSI

Conclusions ICSI

Oocytes activation following ICSI increased the

possibility of parthenogenetic embryo

development.

Need to improve technique to get high diploid

embryos.

8. ICSI

88

SCNT (Cloning)

Cloning procedures

Cytoplast

9. SCNT

Donor cellsNuclear transfer

+_

ET

90

Culture system No. 8-Cell No. (%) embryo developed tocultured Morula Blastocyst

Non co-culture 50 17 (34.0) 10 (20.0) a

Co-cultured with Buff OVD 55 20 (36.4) 17 (30.9) b

Co-cultured with Bovine OVD 55 18 (32.7) 17 (30.9) b

a, b P< 0.05

Development of 8-cell SCNT buffalo embryos derived from fetal fibroblasts:Effects of culture system

Parnpai et al., 1999

9. SCNT

Cloned buffalo embryos

World’s first cloned

buffalo calves birth

91

Shi et al. 2007. Biol. Reprod. 77: 285–291.

Fetal fibroblasts

Pregnant 19% (3/16)

Aborted twin fetuses at day 300

Birth 1, died 20 min.Birth 1, alive

Granulosa cells

Pregnant 20% (1/5)Birth 1, died at 14 d.

9. SCNT

92

Hand made cloning

Birth August 22, 2010

Cloned buffalo in India

National Dairy Research Institute (NDRI)

93

Isolation and establishment of somatic cells from frozen-thawed semen

and use as donor cells for cloning (hand made cloning).

24 cloned embryos ET to 12 recipients, 1 pregnant and gave birth healthy calf.

Cloned buffalo in India

Semen fromthis bull

Cloned calf at 6 months

Selokar et al., 2014

94

Birth October 25, 2011

Cloned buffalo in Thailand

Donor

Tasripoo et al., 2014

High

Low

Gene expression

DNA methylation

DNA methylationSomatic cell cloning

Developmental time

9. SCNT

96

Conclusions SCNT

SCNT still has low birth rate due to high abortion, calf died

before and after birth.

Need to do more study to improve reprogramming of

somatic cells.

9. SCNT

97

Overall conclusions

Efficient production of buffalo calves

Moderate genetics Elite genetics

Ovsynch+FTAI

Recipient in ET program

OPU-IVPSex sorted semen

SCNTMO-ET ?

ICSI ?

Vitrify

oocytes

Genomic

screening

Nutrition, Health & Management

Vitrify embryos

Sexing embryos

98

Acknowledgements

Prof. Pietro Baruselli

University of Sao Paolo, Brazil

state of the art of reproductive biotechnologies in buffaloes4r... · estrus 2-5 days induce...

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