glucose use and lactate production by equine fresh … · 2014. 10. 2. · j. ponthier1,4, p. de...

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J. Ponthier 1,4 , P. de Tullio 3 , S. Parrilla-Hernandez 4 , D. Blommaert 4 , O. Gaspard 2 , S. Deleuze 1,4 1 Equine Clinic, 2 CPMA and 3 CIRM, ULg University of Liège, Liège, Belgium 4 LINALUX-MLS, Centre Européen du Cheval, Vielsalm, Belgium GLUCOSE USE AND LACTATE PRODUCTION BY EQUINE FRESH SEMEN IN HUMAN AND EQUINE EXTENDERS Material and methods Introduction Results Conclusions Human extender doesn’t support equine semen storage: progressive motility is rapidly lower Effect on progressive motility rather than on total: Non-progressive motility previously associated with glycolysis Glucose use and lactate production negligible: No differences between 40 & 100x10 6 spz/ml Glucose increased in INRA96 after 24 hours of storage: Extracellular cleavage of complexe carbohydrates? Raw semen analysis: Concentration Motility (CASA) 20 ejaculates: -5 stallions -4 collections Storage: 24hours, 20°C, tubes closed Motility (CASA): 1, 2, 4, 8, 24 h Nuclear Magnetic Resonance: glucose and lactate concentrations: 24h 40 x 10 6 spz /ml 100 x 10 6 spz /ml 1ml supernatant: ¼ semen ¾ extender 40 x 10 6 spz /ml 100 x 10 6 spz /ml 100 x 10 6 spz ¼ semen ¾ extender 40 x 10 6 spz Centrifugation 1000xg 20 min 100 x 10 6 spz 40 x 10 6 spz ALLGRADWASH INRA96 Statistics: kruskall-wallis test for median differences Aims of the study: to compare equine semen motility, glucose use and lactate production in: Equine Semen Exenders Human Semen Extenders -INRA96 TM : -Milk proteins -Lactose & glucose -No lactate -Allgradwash TM : -No proteins -Glucose no lactose -Lactate 0 20 40 60 80 100 120 0 6 12 18 24 Total motility in INRA 96 Total motility in Human Medium Progressive motility in INRA 96 Progressive motility in Human medium 0 20 40 60 80 100 120 0 6 12 18 24 Total motility in INRA 96 Total motility in Human Medium Progressive motility in INRA 96 Progressive motility in Human medium Preservation of motility at 40x10 6 spz/ml Preservation of motility at 100x10 6 spz/ml Glucose after 24 hours in Allgradwash TM when compared to INRA96 Lactate after 24 hours in Allgradwash TM when compared to INRA96 Median glucose concentration in INRA 96: 17.86mmol in native vs 25.57mmol after 24h of semen preservation % of preservation of motility * * * * * * * * * * * * * Glucose Fructose Active intra-cellular transport Aerobiosis: CO 2 & H 2 O Mito Anaerobiosis: 2 lactate per glucose consumed Non-progressive motility

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  • J. Ponthier1,4, P. de Tullio3, S. Parrilla-Hernandez4, D. Blommaert4, O. Gaspard2, S. Deleuze1,4 1Equine Clinic, 2CPMA and 3CIRM, ULg University of Liège, Liège, Belgium 4LINALUX-MLS, Centre Européen du Cheval, Vielsalm, Belgium

    GLUCOSE USE AND LACTATE PRODUCTION BY EQUINE FRESH SEMEN IN HUMAN AND EQUINE EXTENDERS

    Material and methods Introduction

    Results

    Conclusions • Human extender doesn’t support equine semen storage: progressive motility is rapidly lower  Effect on progressive motility rather than on total: Non-progressive motility previously associated with glycolysis • Glucose use and lactate production negligible:  No differences between 40 & 100x106spz/ml • Glucose increased in INRA96 after 24 hours of storage:  Extracellular cleavage of complexe carbohydrates?

    Raw semen analysis: • Concentration • Motility (CASA)

    20 ejaculates: - 5 stallions - 4 collections

    • Storage: 24hours, 20°C, tubes closed • Motility (CASA): 1, 2, 4, 8, 24 h • Nuclear Magnetic Resonance: glucose and lactate concentrations: 24h

    40 x 10

    6spz /ml

    100 x 10

    6spz /ml

    1ml supernatant: ¼ semen ¾ extender

    40 x 10

    6spz /ml

    100 x 10

    6spz /ml

    100 x 106spz

    ¼ semen ¾ extender

    40 x 106spz

    Centrifugation 1000xg 20 min

    100 x 106spz

    40 x 106spz

    ALLGRADWASH INRA96

    •  Statistics: kruskall-wallis test for median differences

    Aims of the study: to compare equine semen motility, glucose use and lactate production in:

    Equine Semen Exenders Human Semen Extenders

    - INRA96TM: - Milk proteins - Lactose & glucose - No lactate

    - AllgradwashTM: - No proteins - Glucose no lactose - Lactate

    0

    20

    40

    60

    80

    100

    120

    0 6 12 18 24

    Total motility in INRA 96 Total motility in Human Medium Progressive motility in INRA 96 Progressive motility in Human medium

    0

    20

    40

    60

    80

    100

    120

    0 6 12 18 24

    Total motility in INRA 96 Total motility in Human Medium Progressive motility in INRA 96 Progressive motility in Human medium

    Preservation of motility at 40x106spz/ml Preservation of motility at 100x106spz/ml

    • Glucose after 24 hours •  in AllgradwashTM when compared to INRA96

    • Lactate after 24 hours •  in AllgradwashTM when compared to INRA96 •  Median glucose concentration in INRA 96: • 17.86mmol in native vs 25.57mmol after 24h of semen preservation

    % of preservation of m

    otility

    * ** *

    *

    * **

    *

    *

    *

    *

    *

    Glucose

    Fructose

    Active intra-cellular transport

    Aerobiosis: CO2 & H2O

    Mito

    Anaerobiosis: 2 lactate per glucose consumed

    Non-progressive motility