1

Alternative Treatments for Alternative Treatments for SuperovulationSuperovulation of Beef and Dairy of Beef and Dairy

Donor CowsDonor Cows

G.A. BG.A. Bóó11, , R.J. MapletoftR.J. Mapletoft22, , and G.P. Adamsand G.P. Adams22

11Instituto de ReproducciInstituto de Reproduccióón Animal Cn Animal Cóórdoba rdoba (IRAC), C(IRAC), Cóórdoba, Argentinardoba, Argentina

22WCVM, University of Saskatchewan, Saskatoon, WCVM, University of Saskatchewan, Saskatoon, SK, SK, CanadaCanada

Need to increase the supply of transferable Need to increase the supply of transferable embryos, but variability is highembryos, but variability is high……

Embryo transfer Embryo transfer -- SuperovulationSuperovulation

2

Approximately 70% of the variability in Approximately 70% of the variability in superovulatorysuperovulatory response can be response can be related to ovarian status at the time of related to ovarian status at the time of gonadotropingonadotropin treatmenttreatment

MonniauxMonniaux et al., 1983et al., 1983

Development of follicles Development of follicles ≥≥4 mm 4 mm in in cattlecattle

22--wavewave

33--wavewave

00 99--1010 2020

OvulationOvulationLuteal phase

00 88--99 22221616

Luteal phase

FSHFSH

FSHFSH

3

SuperovulationSuperovulation and follicular wave and follicular wave dynamicsdynamics

SuperstimulatorySuperstimulatory treatments must be treatments must be initiated at the time of wave emergence initiated at the time of wave emergence to obtain optimal responseto obtain optimal response

Nasser et al., 1993Nasser et al., 1993

SuperstimulatorySuperstimulatory response and response and wave emergencewave emergence

Initiation ofInitiation of No. of FolliclesNo. of Follicles No. ofNo. ofTreatmentTreatment nn (>7mm)(>7mm) OvulationsOvulations

Day Day --11 1111 22.022.0++3.43.4aa 9.59.5++2.32.3bb

Day 0Day 0 77 20.320.3++3.83.8aa 18.418.4++3.43.4aa

Day +1Day +1 66 7.77.7++3.43.4bb 6.76.7++2.22.2bb

Day +2Day +2 66 7.37.3++2.32.3bb 6.56.5++2.32.3bb

abab P<0.05P<0.05Nasser et al., 1993Nasser et al., 1993

4

SuperstimulationSuperstimulation treatment with treatment with pp--FSH (FSH (FolltropinFolltropin––V*)V*)

Day 10 Day 10 –– 80 mg80 mg i.m.i.m. BidBidDay 11 Day 11 –– 60 mg60 mg i.m.i.m. BidBidDay 12 Day 12 –– 40 mg40 mg i.m.i.m. Bid and PGF (AM)Bid and PGF (AM)Day 13 Day 13 –– 20 mg20 mg i.m.i.m. BidBidDay 14 Day 14 -- A.I. (PM)A.I. (PM)Day 15 Day 15 -- A.I. (AM)A.I. (AM)

**Bioniche Animal HealthBioniche Animal Health

5

Synchronization of follicle wave Synchronization of follicle wave emergence for emergence for superstimulationsuperstimulation

Follicular ablationFollicular ablation –– 1.5 days1.5 days(2 largest follicles) (2 largest follicles)

EstradiolEstradiol –– 4 days 4 days (5 mg E(5 mg E--1717ββ oror 2 mg EB or EV plus P4)2 mg EB or EV plus P4)

GnRH/pLHGnRH/pLH –– 2 days 2 days (considerable variability)(considerable variability)

AblationAblation

~ 1.5~ 1.5..daysdays

Waveemergence

Bergfelt et al., 1994

Folli

cle

Siz

e

DaysDays

Synchronization of follicle wave emergence with follicular ablationSynchronization of follicle wave Synchronization of follicle wave

emergence with emergence with follicular ablationfollicular ablation

FSHFSH

6

SuperstimulationSuperstimulation -- Follicle ablationFollicle ablation

Day 0 Day 0 AM AM -- Ablate 2 largest folliclesAblate 2 largest follicles-- Insert CIDR Insert CIDR

Day 1 Day 1 AM AM -- Initiate FSH treatmentsInitiate FSH treatmentsDay 4 Day 4 AM AM -- PGFPGF

PM PM -- PGF & remove CIDRPGF & remove CIDRDay 6Day 6 PM PM -- AIAIDay 7Day 7 AM AM -- AIAIDay 13Day 13 Embryo collectionEmbryo collection

12

10

8

6

4

LHLHProgestin andProgestin andestradiolestradiol--1717ββ

~ 4~ 4..daysdays

WaveWaveemergenceemergence

Bo et al., 1994

Folli

cula

r dia

met

er (

mm

)Fo

llicu

lar d

iam

eter

(m

m)

DaysDays

Synchronization of follicular wave emergencewith progestin + E-17β

Synchronization of follicular wave emergenceSynchronization of follicular wave emergencewith with progestin + Eprogestin + E--1717ββ

FSH

FSH

7

SuperovulationSuperovulation following treatment following treatment with a with a CIDR plus ECIDR plus E--1717ββ/P4/P4

Day 0 Day 0 AMAM -- CIDR + ECIDR + E--1717ββ/P4/P4Day 4 Day 4 AMAM -- Initiate FSH treatmentsInitiate FSH treatmentsDay 6Day 6 AM AM -- PGFPGF

PM PM -- Remove CIDRRemove CIDRDay 8Day 8 AM AM -- estrusestrus

PM PM -- AIAIDay 9Day 9 AM AM -- AIAIDay 15Day 15 Embryo CollectionEmbryo Collection

SuperovulatorySuperovulatory responses and responses and embryo productionembryo production……

Did not differ between ProgestinDid not differ between Progestin--treated cows treated cows superstimulatedsuperstimulated……

4 days after 4 days after estradiolestradiol treatmenttreatment, ,

1 day after 1 day after follicular ablationfollicular ablation or,or,

8 and 12 days after estrus in8 and 12 days after estrus inControl cowsControl cows

8

EstrusDetection

D-0 D-10

FSH (12/12h)

EstrusDet. + AI

D-13 D-0 D-7

PGF2αPGFPGF22αα

EC1. Traditional superstimulation

PGF2αPGFPGF22αα

2. Synchronized follicle wave emergence

EstrusDetection

D-0 D-4

FSH (12/12h)

EstrusDet.+ AI

D-7 D-8 D-15

ECPGF2αPGFPGF22αα

P4E2+P4E2+P4

3. Synchronized follicle wave emergence and ovulation

EstrusDetection

D-0 D-4

FSH (12/12h)

EstrusDetection

D-7 D-8 D-15

ECPGF2αPGFPGF22αα

P4GnRH orLH + AI

E2+P4E2+P4

0102030405060708090

100

48 60 72 84 96 108 120h post PGF

AIAIAIAI

Chesta et al., 2003

Ovulation time in Ovulation time in superstimulatedsuperstimulated donorsdonors

But, cows with high But, cows with high superovulatorysuperovulatory responses ovulated responses ovulated 12 hours earlier than those with lower responses12 hours earlier than those with lower responses

9

Individual Data Individual Data

0%

20%

40%

60%

80%

100%

48 60 72 84 96 108 120

h post PGF

6 ov 37009 ov 13 9 ov 412 ov 1218 ov 2132 ov 3630 ov 6

Chesta et al., 2003

Fixed-time AI protocols for superstimulated donors

PGF2α

2

6

10

14

Day 8Day 7

mm

Day 0 Day 4

EB + P4 LH

P4FSH(4 days; 12/12h)

FSH(4 days; 12/12h)

P4

LH

FTAI

10

ProgestinProgestin

0 0 4 4 6 7 8 9 6 7 8 9 1515

2,5 2,5 mgmg EE--1717ββ50 50 mgmg P4P4

DayDay 6.5 6.5 -- ControlControlPGFPGF AIAI ECEC

FSH 2x4FSH 2x4

Experimental designExperimental design

0 0 4 4 6 7 8 9 6 7 8 9 1515

ProgestinProgestin

DayDay 6.5 6.5 -- GnRHGnRH2,5 2,5 mgmg EE--1717ββ50 50 mgmg P4P4

PGFPGF AIAIGnRHGnRH

ECEC

ProgestinProgestin

0 0 4 4 6 7 8 9 6 7 8 9 1515

2,5 2,5 mgmg EE--1717ββ50 50 mgmg P4P4

DayDay 6.5 6.5 -- ControlControlPGFPGF AIAI ECEC

FSH 2x4FSH 2x4

FSH 2x4FSH 2x4

PGFPGF

Experimental designExperimental design

11

0 0 4 4 6 7 8 9 6 7 8 9 1515

ProgestinProgestin

DayDay 6.5 6.5 -- GnRHGnRH2,5 2,5 mgmg EE--1717ββ50 50 mgmg P4P4

PGFPGF AIAIGnRHGnRH

ECEC

ProgestinProgestin

0 0 4 4 6 7 8 9 6 7 8 9 1515

2,5 2,5 mgmg EE--1717ββ50 50 mgmg P4P4

DayDay 6.5 6.5 -- ControlControlPGFPGF AIAI ECEC

FSH 2x4FSH 2x4

FSH 2x4FSH 2x4

ProgestinProgestin

2,5 2,5 mgmg EE--1717ββ50 50 mgmg P4P4

PGFPGF AIAI

0 0 4 4 6 7 8 9 6 7 8 9 1515

DayDay 7 7 -- ControlControlECEC

FSH 2x4FSH 2x4

Experimental designExperimental design

0 0 4 4 6 7 8 9 6 7 8 9 1515

ProgestinProgestin

DayDay 6.5 6.5 -- GnRHGnRH2,5 2,5 mgmg EE--1717ββ50 50 mgmg P4P4

PGFPGF AIAIGnRHGnRH

ECEC

ProgestinProgestin

0 0 4 4 6 7 8 9 6 7 8 9 1515

2,5 2,5 mgmg EE--1717ββ50 50 mgmg P4P4

DayDay 6.5 6.5 -- ControlControlPGFPGF AIAI ECEC

FSH 2x4FSH 2x4

FSH 2x4FSH 2x4

ProgestinProgestin

2,5 2,5 mgmg EE--1717ββ50 50 mgmg P4P4

PGFPGF AIAIGnRHGnRH

ProgestinProgestin

2,5 2,5 mgmg EE--1717ββ50 50 mgmg P4P4

PGFPGF AIAI

0 0 4 4 6 7 8 9 6 7 8 9 1515

DayDay 7 7 -- ControlControlECEC

0 0 4 4 6 7 8 9 6 7 8 9 1515

DayDay 7 7 -- GnRHGnRHECEC

FSH 2x4FSH 2x4

FSH 2x4FSH 2x4

Experimental designExperimental design

12

Day 6,5. ControlDay 6,5. GnRH

Day 7. ControlDay 7. GnRH

64

74

84

94

104

hour

s

Synchrony of ovulationSynchrony of ovulation

But, there were no differences in But, there were no differences in superovulatorysuperovulatory responses responses or numbers of transferable embryosor numbers of transferable embryos…… Bo et al., 2006Bo et al., 2006

Day 6.5 Day 6.5Day 6.5 Day 6.5 Day 7 Day 7Day 7 Day 7Control Control GnRHGnRH Control Control GnRHGnRH

CIDRCIDR0 4 6 7 8 9 0 4 6 7 8 9 1515

2.5 2.5 mgmg EE--1717ββ50 50 mgmg P4P4

PGFPGF

ControlControlHeatHeat--WatchWatch

AI 12 & 24 h AI 12 & 24 h afterafter estrusestrus EmbryoEmbryoCollectionCollection

CIDRCIDR

pLHpLH (12.5 (12.5 mgmg LutropinLutropin--V)V)AI 12 AI 12 andand 24 h 24 h laterlater

0 4 6 7 8 9 0 4 6 7 8 9 1515

2.5 2.5 mgmg EE--1717ββ50 50 mgmg P4P4

PGFPGF AIAIpLHpLH

FixedFixed--time AI in practicetime AI in practice

Larkin et al., 2006

FSH 2x4FSH 2x4

FSH 2x4FSH 2x4

EmbryoEmbryoCollectionCollection

13

HeatHeat--Watch Watch -- AI 12 & 24 h after estrus, AI 12 & 24 h after estrus, or FTAI 12 and 24 after or FTAI 12 and 24 after pLHpLH

9.69.6ªª11.611.6ªª21.421.4ªª57.357.3ªª(53(53--62)62)

FTAIFTAI(n=10)(n=10)

7.87.8ªª11.011.0ªª28.528.5ªª58.558.5ªª(51(51--69)69)

Heat Heat Watch Watch (n=10)(n=10)

ViableViableTotal Total OvaOva

No. ovulationsNo. ovulationsOvulation Ovulation Time (h)Time (h)(range)(range)

ProtocolProtocol

Larkin et al., 2006

Results Results -- FTAI FTAI vsvs Heat WatchHeat Watch

0123456789

10

n

04:00p.m.

06:00p.m.

08:00p.m.

10:00p.m.

12:00a.m.

02:00a.m.

time

Time of first AI

Control pLH

Larkin et al., 2006

14

ab P<0,05

Grade 1 & 2FertilizedOva/embryosNIntervalHormone

2,7 ± 0,4 a3,5 ± 0,5 a5,0 ± 0,65212 h

4,0 ± 0,5 b5,0 ± 0,6 b6,2 ± 0,84724h

3,6 ± 0,44,6 ± 0,65,9 ± 0,747pLH

3,1 ± 0,53,9 ± 0,65,2 ± 0,752GnRH

Interval from Progestin Removal Interval from Progestin Removal to to GnRHGnRH or or pLHpLH

* P4 * P4 DevicesDevices werewere removed in removed in allall donorsdonors withwith thethelastlast FolltropinFolltropin--V V treatmenttreatment..

Adapted from Chesta et al., 2006.

PGF

P4 device

E2 + P4

D0AM

D7AM

EmbryoCollection

D15AM or PM

FSH

D6AM

D4D4AMAM 12 h

AIpLH

D8D8AM AM oror PMPM 24 h

AI

P4 deviceremoval

Holstein cows Holstein cows -- FTAIFTAI

15

PGF

P4 device

E2 + P4

D0AM

D7AM

EmbryoCollection

D15AM or PM

FSH

D6AM

D4D4AMAM 12 h

AIpLH

D8D8AM AM oror PMPM 24 h

AI

P4 deviceremoval

Holstein cows Holstein cows -- FTAIFTAI

4.74.7±±0.90.9bb5.45.4±±0.90.95.75.7±±1.01.02424Day 8.5Day 8.5XX--P4 deviceP4 device

2.12.1±±0.50.5aa3.33.3±±0.50.53.83.8±±0.70.72424Day 8Day 8

pLHpLH n Total Fertilized Transferablen Total Fertilized Transferable

Bo et al., 2006Bo et al., 2006abab P<0.01P<0.01

5.95.9±±1.31.36.46.4±±1.41.46.56.5±±1.51.51212Day 8.5Day 8.5Day 7.5Day 7.5

GonadotrophinGonadotrophin treatments must be initiated at the time treatments must be initiated at the time of follicular wave emergence to optimize of follicular wave emergence to optimize superovulatorysuperovulatory responseresponse

Synchronization of follicular wave emergence offers Synchronization of follicular wave emergence offers the convenience of initiating FSH treatments quickly the convenience of initiating FSH treatments quickly and at a selfand at a self--appointed timeappointed time……

Without the necessity of estrus detectionWithout the necessity of estrus detectionWithout sacrificing responseWithout sacrificing response

Evidence suggests that these procedures can be Evidence suggests that these procedures can be incorporated into protocols that involve fixedincorporated into protocols that involve fixed--time AI of time AI of donor cows, without sacrificing ova/embryo qualitydonor cows, without sacrificing ova/embryo quality

Summary

16

PGF2α

P4 device

E + P4

D0 D7PM

D5

EC

D15PM

FSH

D6D4 D9AM

IA

pLH or GnRH 24h

D8.5PM

D9PM

IA

P4 deviceremoval

PGF2α

P4 device

E + P4

D0 D8AM

D5

EC

D16AM

FSH

D6D4D4PMPM

D9PM

IA

pLH or GnRH 24h

D9D9AMAM

D10AM

IA

P4 deviceremoval

17

PGF2α

CRESTAR®

EB (2mg)

D0 D7T

PGF2α

D8T

AI

EC

D5 D15M

GnRH 12h

FSH

D6D4 D8M

D9M

AI

XCRESTAR®

CRESTAR®

EB (2mg)

D0 D7T

D8T

AI

EC

D5 D15M

GnRH 12h

FSH

D6D4 D8M

D9M

AI

Retirada doCRESTAR®

0.710.710.810.810.280.288.40 8.40 ±± 1.40 1.40 9.90 9.90 ±± 0.780.78Total Ova/Total Ova/embryosembryos (n)(n)

0.880.880.880.880.030.034.20 4.20 ±± 0.740.746.80 6.80 ±± 0.660.66TransferableTransferable EmbryosEmbryos (n)(n)

0.990.990.430.430.030.033.50 3.50 ±± 0.650.655.90 5.90 ±± 0.710.71FreezableFreezable EmbryosEmbryos (n)(n)

50.050.068.768.7 0.010.01

TratTrat

0.540.540.680.68PercentajePercentaje ofof ViableViable EmbryosEmbryos(%)*(%)*

TratTrat vsvsRepRepRepRep

PPSexedSexedSemenSemen(n=10)(n=10)

NonNon SexedSexedSemenSemen(n=10)(n=10)

BaruselliBaruselli et al., 2007et al., 2007

18

68,7

50.0

34,4

45.0

0

10

20

30

40

50

60

70

Taxa

de

embr

iões

viá

veis

(%)

embriões ♀ e ♂ embriões ♀ ou ♂

Convencional Sexado

(68/99) (42/84) (34/99) (38/84)(68/99) (42/84) (34/99) (38/84)

aa

bb

Total de embriões

BaruselliBaruselli et al., 2007et al., 2007

Repeated superstimulation - E-17ββDay 0 Day 0 -- Insert CIDR Insert CIDR

-- inject 5 mg Einject 5 mg E--1717ββ plus 100 mg P4plus 100 mg P4Day 4 Day 4 -- Initiate FSH treatmentsInitiate FSH treatmentsDay 6 Day 6 -- PGFPGF

-- PGF & remove CIDRPGF & remove CIDRDay 8Day 8 -- AIAIDay 9Day 9 -- AIAIDay 15 Day 15 -- Embryo collection & transferEmbryo collection & transfer

-- PGFPGFDay 22 Day 22 -- Estrus Estrus -- PGFPGFDay 30Day 30 -- Insert CIDR Insert CIDR

-- inject 5 mg Einject 5 mg E--1717ββ plus 100 mg P4plus 100 mg P4

19

34.1134.1130.230.239.239.2Flush intervalFlush interval13421342893893449449Total # 1 collectTotal # 1 collect

8.58.5Traditional 60 dayTraditional 60 dayTransfer/flushTransfer/flush(1038 Donors)(1038 Donors)

8.18.18.68.67.27.2Transfer/FlushTransfer/Flush(IETS #1 & #2)(IETS #1 & #2)

0.930.931.01.00.80.8Avg. # 2/FlushAvg. # 2/Flush7.147.147.67.66.46.4Avg. # 1/FlushAvg. # 1/Flush

3.493.493.93.93.03.0Avg. # of FlushesAvg. # of Flushes1881881181187070# of Flushes# of Flushes535330302323# of Donors# of Donors

PooledPooledRed AngusRed AngusHolsteinHolstein

Repeated Repeated superovulationsuperovulation –– EE--1717ββ

Stables, 2003Stables, 2003

GnRH/pLH(Ovulation?)

~2.0.days

Waveemergence

Martinez et al., 1999

Folli

cle

Size

Days

Follicle wave emergence - GnRH or pLHFollicle wave emergence Follicle wave emergence -- GnRHGnRH or or pLHpLH

FSH

20

The problemThe problem……

GnRHGnRH or or pLHpLH will only synchronize will only synchronize follicle wave emergence if ovulation is follicle wave emergence if ovulation is induced. However,induced. However,……

Day of follicular wave emergence Day of follicular wave emergence following following GnRHGnRH or or pLHpLH

Martinez et al., 1999

DayDay --22 --11 TxTx +1+1 +2+2 +3+3 +4+4 +5+5 +6+6

Day 3Day 3OvOv (14)(14) 1010 11 11 22NoNo (4)(4) 11 22 11

Day 6Day 6OvOv.. (14)(14) 11 1111 22NoNo (4)(4) 33 11

Day 9Day 9OvOv.. (8)(8) 11 77NoNo (10)(10) 22 22 44 11 11

OverallOverallOvOv.. (36)(36) 22 2828 33 11 2 2 NoNo (18)(18) 22 22 44 44 33 22 11

21

FA/EFA/E--1717ββ pLH/GnRHpLH/GnRHnn 5454 5151No. of CLNo. of CL 20.120.1 19.719.7Total ova/embryos Total ova/embryos 8.08.0 4.94.9

(%) (%) (40)(40)aa (25)(25)bb

Fertilized Ova Fertilized Ova 4.54.5 2.62.6(%) (%) (56)(56) (53)(53)

Trans. Trans. EmbEmb.. 3.33.3 2.02.0(%) (%) (41)(41) (41)(41)

SuperovulatorySuperovulatory response in Holstein cows after response in Holstein cows after follicular ablation follicular ablation (FA)(FA)//EE--1717ββ or or pLH/GnRHpLH/GnRH

Mitchell et al., 1998 & Deyo et al., 2001(Combined)

abab (P < 0.05)(P < 0.05)

The dilemmaThe dilemma……

Follicle ablation is not readily accessible or Follicle ablation is not readily accessible or easily done in the fieldeasily done in the field

EstradiolEstradiol is not available in many countriesis not available in many countries

GnRHGnRH or or pLHpLH is still not proven, butis still not proven, but……

22

Days 8Days 8--1313 E17E17ββ E17E17ββ** GnRHGnRH

nn 15651565 663663 5656 9595Total ova/embryosTotal ova/embryos

9.99.9 12.412.4 13.313.3 11.211.2Transferable embryosTransferable embryos

5.4 5.4 6.26.2 5.45.4 5.55.5(%) (%) (54)(54) (50)(50) (41)(41) (50)(50)

SuperovulatorySuperovulatory response response -- Holsteins Holsteins Days 8Days 8--13 or 4 d after E13 or 4 d after E--1717ββ or 2.5 d after or 2.5 d after GnRHGnRH

Steel, unpublished* Last year * Last year –– Sexed semenSexed semen

HinshawHinshaw and Beal, 2007and Beal, 2007

23

PGF2α

CIDR or Cue-Mate

D0 D7AM

D5

EC

D15

FSH

D6D4 D8PM

IA

pLH 24h

D8AM

D9AM

IA

P4 deviceremoval

D2

GnRH orpLH

PGF PGF GnRH PGF

140 28 35 37

14 days 12 days 7 days 2 days

GnRH AI

12 h

Fricke, 2005

Days

Presynch

PresynchronizationPresynchronization may improve response by may improve response by ensuring the presence of an ensuring the presence of an ovulatoryovulatory--sized sized follicle at the time of follicle at the time of GnRHGnRH treatment!!treatment!!

24

AlternativesAlternatives……Disregard wave status Disregard wave status Employ Employ ultrasonographyultrasonography to determine the to determine the emergence of a waveemergence of a waveSuperstimulationSuperstimulation on the first follicle waveon the first follicle waveDownDown--regulate the pituitary glandregulate the pituitary glandSubordinate follicle breakSubordinate follicle break--throughthrough……(recruit a new wave with (recruit a new wave with eCGeCG or FSH at or FSH at random stages of the cycle) random stages of the cycle)

Disregard wave statusDisregard wave statusset-up day VS viable embryos

0123456789

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 C

set-up day

viab

le e

mbr

yos

MacDonald, unpublishedMacDonald, unpublished

25

Singh et al., 2003Singh et al., 2003

UltrasonographyUltrasonography to determine emergence of a to determine emergence of a follicle wave follicle wave –– Follicle numbersFollicle numbers……

Number of follicles at end of Number of follicles at end of superstimulatorysuperstimulatorytreatment correlated to number of follicles treatment correlated to number of follicles recruited into the same or the preceding waverecruited into the same or the preceding wave

Simple U/S test will Simple U/S test will permit:permit:

1.1. Determine FWE, Determine FWE, 2.2. Prediction of likely Prediction of likely

donor response, donor response, 3.3. Selection of high Selection of high

producing donors, producing donors, 4.4. Prediction of Prediction of

recipient needsrecipient needs

SuperstimulationSuperstimulation on the first waveon the first wave(Wave 1 (Wave 1 vsvs Wave 2)Wave 2)

Treatment groupTreatment groupEnd Point * End Point * Wave 1Wave 1 Wave 2Wave 2

No. of heifersNo. of heifers 1919 2121Ovulations (U/S)Ovulations (U/S) 6.66.6++1.01.0 8.28.2++1.71.7No. of CL (slaughter)No. of CL (slaughter) 6.56.5++0.90.9 8.18.1++1.81.8Total ova/embryos Total ova/embryos 5.35.3++0.70.7 5.35.3++0.90.9Fertilized ovaFertilized ova 3.03.0++0.60.6 3.33.3++0.70.7*no significant difference*no significant difference

Adams et al., 1994Adams et al., 1994

26

Superovulation: First follicle wave

Day 0 -EstrusDay 1 -CIDR – Folltropin BidDay 2 -Folltropin BidDay 3 -Folltropin BidDay 4 -Folltropin Bid –Day 5 - Folltropin Bid + PGF – AM and PM

Remove CIDR AMDay 6 - LutropinDay 6 PM AIDay 7 AM AIDay 13 - Embryo Collection

2.52.5±±1.21.22.5 2.5 ±± 1.61.67.07.0±±2.72.7UnfertilizedUnfertilized ovaova

5.15.1±±1.71.7bb5.8 5.8 ±± 1.41.4bb0.0 0.0 ±± 0.00.0aaFreezableFreezable embryosembryos

6.66.6±±2.02.0bb8.0 8.0 ±± 1.81.8bb0.2 0.2 ±± 0.20.2aaTransferableTransferable embryosembryos

9.29.2±±2.32.311.7 11.7 ±± 2.92.98.3 8.3 ±± 2.92.9Total ova/Total ova/embryosembryos

14.5 14.5 ±± 2.52.512.7 12.7 ±± 2.72.715.3 15.3 ±± 4.34.3CLCL

23.023.0±±3.73.718.5 18.5 ±± 4.54.521.521.5±± 6.66.6FolliclesFollicles > 8 mm > 8 mm atat FTAIFTAI

666666NN

EB+P4EB+P4PlusPlus CIDRCIDR

FirstFirst WaveWavePlusPlus CIDRCIDR

FirstFirst WaveWaveNo CIDRNo CIDR

GroupsGroups

(a,b P<0.05)Nasser et al., 2002 (IETS)

SuperstimulationSuperstimulation of the of the first first wavewave

27

CUEMATE

PGF GnRHCUEMATE

FSH

PGF Day 4 PM and Day 5 AM

pLH AI AIEmbryo collect

PGF

D-3 PM D7AMD-13 PM D0 PM D4 PM D5AM D6AM D6 PMD-1 AM D13AM

SuperstimulationSuperstimulation -- First follicular waveFirst follicular waveThe setThe set--upup

Carballo GuerreroCarballo Guerrero and Band Bóó, 2007, 2007

00 1010 1313 1818 2020 2626

GnRH

OvulationOvulation

FSH

CueCue--MateMate

FSH

CueCue--MateMatePGFPGFPGFPGF

28

Superovulatory response in Bonsmara cows and heifers treated with Folltropin-V during the first follicular wave or

4 days after estradiol (Control)

3.8 ± 0.73.3 ± 0.65.0 ± 0.99.1 ± 1.239Heifers

5.1 ± 1.04.3 ± 1.06.7 ± 1.410.7 ± 1.629Cows

3.7 ± 0.83.0 ± 0.65.2 ± 1.18.4 ± 1.434Wave 1

5.1 ± 0.94.5 ± 0.96.3 ± 1.111.0 ± 1.434Control

Grades 1, 2 & 3 embryos

Grades 1 & 2 embryos

Fertilized ova

Total ova and embryos

nMain effects

Means did not differ (P>0.20)Carballo GuerreroCarballo Guerrero and Band Bóó, 2007, 2007

Cue-Mate

PGF2α GnRH

Cue-Mate

FSH

PGF D4 PM and D5 AM

pLH AI AIEmbryo Collection

PGF2α

D-3PM

D7AM

D-8 PM D0 PM D4 PM D5AM

D6AM

D6 PMD-1 AM

D13AM

29

8.2 ± 3.79.4 ± 3.612.9 ± 4.711.0 ± 2.39Control

6.1 ± 3.59.0 ± 3.512.3 ± 3.815.6 ± 2.08First Wave

Transferable embryos

Fertilized ova

Total ova/embryos

CLNGroup

Means did not differ (P > 0.4).Bo et al., 2008

2

4

6

12

1

Folli

cle

s ize

(mm

)

Day 0 2 3 4 5 6

1010

88

FSHFSH

Follicle breakFollicle break--throughthrough

CIDRCIDR

500 IU500 IUeCGeCG

30

2

4

6

12

1

Folli

cle

s ize

(mm

)

Day 0 2 3 4 5 6

1010

88

FSHFSH

Follicle breakFollicle break--throughthrough

CIDRCIDR

500 IU500 IUeCGeCG

FSHFSH

SuperovulatorySuperovulatory response in response in AngusAngus embryo embryo donorsdonors withwith historyhistory ofof poorpoor response response

EffectEffect ofof pretreatmentpretreatment withwith eCGeCG

Total Total FertFert GradesGrades Grades Grades TxTx nn CL CL ova/ova/embemb ovaova 1+21+2 1+2+31+2+3

No No eCGeCG 2626 8.58.5±±1.11.1 3.13.1±±0.70.7 1.51.5±±0.40.4 1.01.0±±0.30.3 1.01.0±±0.20.2

eCGeCG 2626 12.012.0±±1.31.3 7.77.7±±1.51.5 4.44.4±±0.70.7 3.53.5±±0.60.6 3.63.6±±0.60.6

PP--valuevalue 0.0050.005 0.0070.007 0.00050.0005 0.00030.0003 0.00020.0002

Carballo GuerreroCarballo Guerrero et al., 2007et al., 2007

31

2

4

6

12

1

Folli

cle

s ize

(mm

)

Day 0 1 2 3 4 5 6

1010

88

FSH

FSHFSH

Standard treatmentStandard treatment

CIDRCIDR

2

4

6

12

1

Folli

cle

s ize

(mm

)

Day 0 1 2 3 4 5 6

1010

88

FSH

FSHFSH

Follicle breakFollicle break--throughthrough

CIDRCIDR

32

PGF2α

Cue-Mate

D0 D7AM

D5

EC

D15

Folltropin-V

D6D4 D8PM

IA

pLH 24h

D8AM

D9AM

IA

P4 deviceremoval

D2

4.9 ± 1.7(n = 18)

6.1 ± 2.0(n = 15)

10.5 ± 2.3(n = 15)

14.1 ± 1.2(n = 26)

Control

3.6 ± 1.0(n = 16)

4.7 ± 1.1(n = 16)

6.9 ± 1.3(n = 16)

12.3 ±1.1(n = 29)

6-d FSH

Transferable embryos

Fertilized ova

Total ova/embryosCLGroup

Superovulatory response in embryo donorstreated with FSH at random stages of the cycleControl - (for 4 days following EB plus P4 device) 6-d FSH - (for 6 days following P4 device)

33

Repeated Superstimulation without E-17ββDay 0 Day 0 -- Insert CIDRInsert CIDRDay 2 Day 2 -- Initiate FSH preInitiate FSH pre--treatmentstreatmentsDay 4 Day 4 -- Initiate FSH Initiate FSH superstimulatorysuperstimulatory treatmentstreatmentsDay 6Day 6 -- PGFPGF

-- PGF & remove CIDRPGF & remove CIDRDay 8 PM Day 8 PM -- AIAIDay 9 AM Day 9 AM -- AIAIDay 15 Day 15 -- Embryo collection & transferEmbryo collection & transfer

-- PGFPGFDay 22 Day 22 -- Estrus Estrus -- PGFPGFDay 28Day 28 -- Insert CIDR Insert CIDR Day 30Day 30 - Initiate FSH pretreatments

SummarySummaryFSH treatments must be initiated at time of follicle wave FSH treatments must be initiated at time of follicle wave emergence to optimize emergence to optimize superovulatorysuperovulatory responseresponse

Follicle ablation or Follicle ablation or estradiolestradiol will synchronize follicle wave will synchronize follicle wave emergence effectively for emergence effectively for superstimulationsuperstimulation

An effective protocol with An effective protocol with GnRHGnRH may be availablemay be available

SuperstimulationSuperstimulation can be initiated effectively at the time of can be initiated effectively at the time of emergence of the first follicle wave (day of ovulation)emergence of the first follicle wave (day of ovulation)

Follicle breakFollicle break--through has the potential to be used in through has the potential to be used in superstimulationsuperstimulation protocols avoiding the need to synchronize protocols avoiding the need to synchronize follicle wave emergencefollicle wave emergence

34

Photo AlbumPhoto Album

by rjm404by rjm404

Thank youThank you

Muchas Gracias

Clinica do Leite – ESALQ/USP

Uso de sêmen sexado paraSOTF em gado de corte

Projeto em parceria com:• Sexing• Lagoa da Serra• Intervet• Departamento de Reprodução Animal, FMVZ-USP

35

Fixed-time AI protocols for superstimulated Bos indicus donors

PGF2α

2

6

10

14

Day 8Day 7

mm

Day 0 Day 4

EB + P4 LH

P4FSH(4 days; 12/12h)

FSH(4 days; 12/12h)

P4

LH

↑E2 + Estrus behaviour

FTAI

0102030405060708090

0 12 24 36 48 60 72 84 96 108 120Hours after PGF

% o

f ovu

latio

nsP12/LH48P24/LH48P36/LH48

PGF(Day 6 AM)

P4 (24h)

P4 (36h)

FSH (4 Days;12/12h)

P4 (12h)

LH(Day 8 AM)

Distribution of ovulations (indicated in hours after the first PGF) in superstimulated Nelore (Bos indicus) donors treated with progesterone releasing devices (DIB) for 6.5d (P12), 7d (P24) or 7.5d (P36) with LH treatment on Day 8

Ultrasonography (12/12h)

Martins et al., 2005

P12 vs P24 = >0.05P12 vs P36 = <0.05P24 vs P36 = <0.05

36

Grupo sêmen convencional (n=10)

PGF2α

CRESTAR®

BE (2mg)

D0 D7T

PGF2α

D8T

IA

Colheita de

Embrião

D5 D15M

GnRH 12h

FSH

D6D4 D8M

D9M

IA

Retirada doCRESTAR®

Grupo sêmen sexado (n=10)

CRESTAR®

BE (2mg)

D0 D7T

D8T

IA

Colheita de

Embrião

D5 D15M

GnRH 12h

FSH

D6D4 D8M

D9M

IA

Retirada doCRESTAR®

Experimental design1st Replicate 2nd Replicate(35d)

Group P24/LH48

Group P24/LH60

Sêmen sexado(n=5)

Sêmen convencional (n=5)

Sêmen sexado(n=5)

Sêmenconvencional (n=5)

The same sires were used in all replicates

37

Tabela - Taxa de prenhez aos 30 e 60 dias após a transferência de embriões provenientes de inseminação com sêmen convencional e sexado e porcentagem de fêmeas na sexagem fetal.

90,0 (18/20)47,6 (20/42)57,1 (24/42)42Sêmen sexado

52,7 (19/36)52,9 (36/68)57,3 (39/68)68Sêmen convencional

Sexagem fetal (% de fêmeas)

Taxa de prenhez60 dias (%)

Taxa de prenhez30 dias (%)NEmbriões

produzidos