1 Alternative Treatments for Alternative Treatments for Superovulation Superovulation of Beef and Dairy of Beef and Dairy Donor Cows Donor Cows G.A. B G.A. Bó 1 , , R.J. Mapletoft R.J. Mapletoft 2 , , and G.P. Adams and G.P. Adams 2 1 Instituto de Reproducci Instituto de Reproducció n Animal C n Animal Có rdoba rdoba (IRAC), C (IRAC), Có rdoba, Argentina rdoba, Argentina 2 WCVM, University of Saskatchewan, Saskatoon, WCVM, University of Saskatchewan, Saskatoon, SK, SK, Canada Canada Need to increase the supply of transferable Need to increase the supply of transferable embryos, but variability is high embryos, but variability is high… Embryo transfer Embryo transfer - Superovulation Superovulation
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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
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
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
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
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
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……
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Days 8Days 8--1313 E17E17ββ E17E17ββ** GnRHGnRH
nn 15651565 663663 5656 9595Total ova/embryosTotal ova/embryos
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
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
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
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.