publication barcelona eaap 2009
TRANSCRIPT
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A new freezing extender for stallion semen
to get high fertility rates INRA-Freeze®
Elodie Pillet, G. Duchamp, F. Batellier, J.M. Yvon, G. Delhomme, S. Desherces, E. Schmitt, M. Magistrini.
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BUT
Artificial insemination with frozen semen
A lot of advantages
A few limits
Transport of semen is easier
Storage can be unlimited
Choice of stallion is wider
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Results are lower or more fluctuating
Freezing extenders are not optimal, and not practical
Have to be prepared in the lab
Contain milk and/or egg yolk
than with fresh or chilled semenIn France, in the National Studs: 2007: Fertility/cycle = 44%44% (2557 cycles) 2008: Fertility/cycle = 47%47% (2270 cycles)(55% with fresh semen in the National Studs)
For the success of artificial insemination
Freezing extender = a key factor
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Objective :
To develop a freezing extender
able to improve fertility results
easy to use
able to avoid sanitary risks
in vitro analyses + in vivo trials 2 steps : ► 1) To remove milk
► 2) To remove egg yolk (EY)
2 in vivo trials
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INRA - Nouzilly
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Experimental herd : 120 mares
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► 1) To remove milkFertility Trial 1
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1st dilution
2nd dilution and freezing
INRA82+ 2% EY
+ 2,5% glycerol
INRA82
INRA96®
(only caseins of milk)
+ EY + glycerol
INRA96®
+ 2% EY+ 2,5% glycerol
INRA96®(Palmer, 1984) (Batellier et al., 1997)
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Insemination Protocol :
induction of ovulation (CEG)
1 dose of insemination
400 x 106 sperm cells
6 hours before expected ovulation
insemination in the uterine body
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0
20
40
60
80
100
Stallion 1 Stallion 2 Stallion 3
3 stallions, 7 ejaculates/stallion
84 mares’ cycles inseminated (72 mares)
INRA82 + EY + glycerol << INRA96® + EY + glycerol
0
20
40
60
80
100
Per-cycleFertility
(%)
p<0.01
71%30/42
INRA82+egg yolk+glycerol
INRA96®
+egg yolk+glycerol
40%17/42
(Pillet et al., 2008)
(milk based) (only caseins)
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► 2) To remove egg yolkFertility Trial 2
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plasma
granules
1) Contains Low DensityLipoproteins LDL
2) Can be sterilized
Sterilized Egg yolk plasma
Fractions of egg yolk
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1st dilution
2nd dilution and freezing
INRA96®
+ 2% EY+ 2,5% glycerol
INRA96®
INRA96®
+ Sterilized EY plasma+ glycerol
INRA96®
+ 4% ster. EY plasma+ 2,5% glycerol
INRA96®
Insemination
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70 mares’ cycles inseminated (68 mares)
0
20
40
60
80
100
p>0.05
Per-cycleFertility
(%)60%21/35
69%24/35
INRA96®
+EY+glycerol
INRA96®
+sterile EY plasma+glycerol
2 stallions, 8 ejaculates/stallion
0
20
40
60
80
100
Stallion 1 Stallion 2
INRA96® + EY + glycerol = INRA96® + + glycerolSterilizedEY plasma
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Conclusion :
Fertility was greatly improved using INRA96® + egg yolk + glycerol OR
INRA96® + sterilized egg yolk plasma + glycerol
Can be incorporated into a ready to use extender
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2 steps to develop a new freezing extender
INRA-Freeze®
2
1
2
INRA-Freeze®
Increases fertility results Is ready to use Avoids sanitary risks
69%69%(65% with fresh semen with
the same experimental herd at INRA)
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Perspectives
CH3
lCH
3 – N + - l
CH3
C
H 3
l
CH 3 – N
+ - C 2 H 4 – OH
l
C
H 3
CH3
l
CH3 – N
+ - C2
l
CH3
Which molecule(s) ?
apoproteins
phospholipids
Which phospholipid ?
Which mechanism ?
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INRA82 > INRA96 ®
INRA82+EY+GINRA96®+EY+G
Valeurs moyennes
0
20
40
60
80
100
VAP(µm.s-1) % PROG % RAP Paramètres de mobilité
****
******
INRA96 ® > INRA82
12 / 35 / 63 / 103 / 154 / 205 / 303 mOsm30
40
50
60
70
80
90
100% SPZ aux membranes
endommagées (marquage IP+)
INRA82+JO+GINRA96®+JO+G*% rapides : 61% > 58% (p<0,01)
Motility Membrane integrity
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0
20
40
60
80
100
INRA82
spz à acrosomes intacts(%)
a
****
b
INRA96®
Resistance to oxidative stress
INRA82 > INRA96 ®INRA96 ® > INRA82 INRA82 = INRA96 ®
PSA-FITC
69% > 51% (p<0,0001)0,59 > 0,52 (p<0,0001)
Calcul du ratio :
fluo verte
(fluo rouge + verte)
020406080
100120140160180200
intensité de fluorescence FilipinIII
INRA82 INRA96®
aa
Non significatif
C11-Bodipy
Filipin III
Acrosome integrity
Cholesterol content
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Motility results:Motility results:IVOS
0
10
20
30
40
50
60
70
80Valeursmoyennes
VAP (µm/s) % PROG % RAP30 % RAP40
a b
a a
a a a a
a,b : p < 0.001
Egg yolk
Plasma