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Yangqing LuGuangxi University, China

2017.11.16 Chitwan Nepal

Milk Consumption per capita

Country Milk Consumption (kg/capita/yr)

Australia 230.92

USA 253.8

India 68.72

China 28.7

World Average 108

http://chartsbin.com/view/1491

Distribution of dairy cows in China

Distribution of dairy cows Distribution of buffaloes(Colored province)

Milk Production

FAO2009 http://chartsbin.com/view/1482

Buffalo Stock

http://chartsbin.com/view/33407

Country Buffalo (head)Total Milk Production

(ton)

India 115,420,000 62,860,000

Pakistan 33,700,000 21,622,000

China 23,250,000 3,000,000

Egypt 4,200,000 2,702,960

�Mostly Swamp buffaloes!�Small body size and low milk production!�13% of the world stock, but 3% milk productionBuffalo in China

Use Reproductive Biotechnologies to

Improve Buffalo Productivity

� Artificial Insemination

� IVEP and Embryo Transfer

� Sex�presection

� Somatic Cell Nuclear Transfer/Cloning

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The first choice for breeding in many domestic animals for genetic improvement. Small body size and low milk production!Crossbreeding through AI to improve

the milk production

River buffalo

Swamp buffalo

Crossbred

AIMurrahMurrah

19571957

NiliNili--RaviRavi19751975

MediterraneanMediterranean2011, 20142011, 2014

River Buffalo Introduced into China

Guangxi Buffalo Research Institute, China

� Home to more than 1000 head of river buffaloes.

Provincial Breeding Station, Guangxi, China

� Home to over 100 heads of selected bulls.

� Providing 1,000,000 straws of frozen semen annually.

� Distribute to county-level breeding station, FOR FREE!

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Breeding system in Guangxi, China

Small herd size in countryside, mostly swamp buffaloes

Technicians(Breeding station in town or village)

Milk Production in Crossbred Buffalo

18002000

800

1423

1994

0

500

1000

1500

2000

2500

Murrah Nili-Ravi Swamp SxM F2 SxN F2

Milk yield per lactation (kg)

Moioli and Borghese 2005; Yang et al 2007.

Application of deep AI at natural estrus

Deep AIConvention AI

Improvement of conception rate with

application of Deep AI 2008-2013

50.3%

50.8%

51.6%

53.7%

54.3%

55.0%

50.0%

51.0%

52.0%

53.0%

54.0%

55.0%

56.0%

2007 2008 2009 2010 2011 2012 2013 2014

Pregnancy rate of AI in 2008-2013 in Guangxi, China.

Estrus synchronization in buffalo（CIDR/PGF2α）Breed Number Estrous rate Conception rate

Swamp 102 74.5%(76)c 30.4% (31)c

CrossbredF1 326 77.9%(254)b 41.3%(105)b

Murrah 129 86.8%(112)a 47.3%(53)a

Nili-Ravi 98 86.7%(85)a 45.9%(38)a

a,b,c: P<0.05

Considering the low efficiency, estrus synchronization and timed AI is not commercially practical in swamp buffaloes.梁贤威等《畜牧与兽医》 2007.

IVF

OPU

ET

An efficient strategy to expand the herds of high genetic value.

Surrogate（Swamp buffalo)

Female(River buffalo)

Male(River buffalo)

River buffalo calf

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Repeated OPU

(Liang et al., 2007)

OPU was performed on 40 Nili-Ravi and Murrah buffaloes for consecutive 3 times each, at an interval of 3 days.

Oocyte Recovery

Repeated OPU

OPU-IVF Blastocyst Development

(Liang et al., 2007)

Embryo transferEmbryo Recipient No. Recipient Pregnancy (%)

FreshSwamp 51 19 (38.0%)

36 (44.4%)River 31 17(54.8%)

FrozenSwamp 15 3(20.0%) 8 (32.0%)

River 10 5(50.0%)

*Two embryos per recipient. (Li et al., 2012)

� Pre-selection for desired sex of the offspring in animals where productivity depends on sex.

� Rapidly expand the herds by producing more females.

Bull

Cows

Sperm

IVF

Flow Sorting

DNA difference

Sexed Calf

ET

AI

Difference in DNA content between XY sperm

Cytometry detection of the DNA

content of X- and Y-sperm of buffalo

Y X

MalesDNA difference (%，n=6)

Murrah Nili-Ravi

Breed mean* 3.59 ± 0.11 3.55 ± 0.14

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Y YX X

Holstein Buffalo

More overlap is seen in histogram distributions of X- and Y-sperm from river buffalo than Holstein due to the small difference in their DNA content.

~4.0% ~3.6%

Sorting purity and speed

Histogram showing the sorted buffalo Y-sperm enriched sample.

Purity: ~90%

Speed:• Moflo SX: ~5000 sperm/sec

IVF with sexed buffalo spermSperm Cleavage % Blastocyst %Sexed 51 15.3Unsexed 51 19.1P>0.05 (Liang et al., 2008)

OPU-IVF sexed buffalo calfFirst report sexed buffalo calf(Liang et al., 2008)(Lu et al., 2007)

Effect of bulls in AI

BullInsemination

ConceptionCalving

Total

Female

n %P-

value*n %

P-

value*

Murrah 1,706 895 52.5%a 883 777 88.0%

Nili-Ravi 2,815 1,299 46.1%b<0.01

1,280 1,118 87.3%0.65ConceptionConceptionConceptionConceptionBullsBullsBullsBulls InseminationInseminationInseminationInsemination nnnn %%%%Murrah 759 508 66.9%aNili-Ravi 399 233 58.4%b

Sexed semen

Non-sexed semen

Effect of cows AI with sexed semen:

VariableInseminat

ion

ConceptionCalving

Total

Female

n %P-

value*n %

P-

value*

Cow

0.11 0.65Swamp type 4,038 1,984 49.1% 1,963 1,726 87.9%

Crossbred F1 272 143 52.6% 139 119 85.6%

Crossbred F2 68 41 60.3% 41 35 85.4%

Parity of Cow

0.717 0.18

0 197 96 48.7% 93 86 92.5%

1-2 1,765 844 47.8% 835 726 86.9%

3-4 1,534 762 49.7% 756 667 88.2%

>5 517 246 47.6% 243 205 84.4%

Seasonal Effect AI with sexed semen

Season Insemination

Conception

Calving

Total

Female

n % P-value* n % P-value*

Spring(Mar-May) 794 337 42.4%a

<0.01

335 288 86.0%

0.52Summer(Jun-Aug) 789 418 53.0%b 415 362 87.2%

Autumn(Sep-Nov) 1,347 698 51.8%b 684 609 89.0%

Winter(Dec-Feb) 1,591 741 46.6%a 729 636 87.2%

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Seasonal Effect AI with sexed semen

A polynomial trend line fitted to the month's data shows the high conception rate is expected from late summer to early winter, a period of daylight decreasing (dash line).

Optimum breeding season

Co

nce

pti

on

Rat

e

Effect of technicians AI with sexed semen

a,b,c: P<0.01

Co

nce

pti

on

Rat

e

• Copying animal with high genetic value.

• Modify important genes or introduce exogenous genes of interest.

River buffalo Fibroblast (Donor)

Embryo transfer

Enucleated oocytes

River buffalo calves

Reconstructed embryos

Swamp buffalo

Inter-subspecies SCNT

(Yang et al., 2010)

Effects of Scriptaid on the Histone Acetylation

and DNA Methylation on SCNT embryos

(Sun HL, et al 2015)

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Cloned buffalo calvesCloned buffalo calves

First SCNT buffalo(Shi et al., 2007)

Transgenic SCNT buffalo(Lu et al, Trans Res, 2011)

Inter-subspecies SCNT(Yang et al., 2010)

� John Williams et al. Genome assembly and transcriptome resource for river buffalo, Bubalus bubalis (2n = 50). GigaScience, 2017.

� Brigitte Glanzmann et al. The complete genome sequence of the African buffalo (Syncerus caffer). BMC Genomics. 2016

� Lal Teer Ltd. 2014

� Ongoing in China…

SummarySummary� >700,000 AI service are performed each year in China and

the milk and meat production has significantly improved.

� Sex semen is readily for use.

� OPU-IVF has been used in production of buffaloes with high genetic value.

� Emerging biotech as SCNT, GM and Genome Sequencing is under research.

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