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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.
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
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
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
INRA - Nouzilly
Experimental herd : 120 mares
► 1) To remove milkFertility Trial 1
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)
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
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)
► 2) To remove egg yolkFertility Trial 2
plasma
granules
1) Contains Low DensityLipoproteins LDL
2) Can be sterilized
Sterilized Egg yolk plasma
Fractions of egg yolk
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
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
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
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)
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 ?
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
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
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
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