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A flow-cytometric study on the effect of myeloperoxidase on stallion spermatozoal motility and structure
6th ISSR Abstracts / Journal of Equine Veterinary Science 32 (2012) 475-518
stallion's reproductive tract. In particular, the expression of EQ-12, CD52 and CRISP1 is concentrated in the epididymis. These proteins therefore represent suitable candidates for studies into the mechanisms of protein transfer between the epididymis and spermatozoa during the process of equine sperm maturation.
A flow-cytometric study on the effect of myeloperoxidase on stallion spermatozoal motility and structure J. Ponthier 1, 2, S. Parrila-Hernandez 1, 2, F. Van Den Berghe 1, M. Savic 3, and S. Deleuze 1, 2 1 Equine Clinic, Veterinary Medicine Faculty, Center, ULg University of Liège, B-4000, Liège, Belgium, 2 LINALUXMLS, Centre Européen du Cheval, 18 rue des Champs Elysées, B-5590, Ciney, Belgium, 3 IMV Technologies, ZI n 1 Est, Laigle, F-61300, L'aigle, France Myeloperoxidase (MPO) is a pro-oxidant enzyme that is normally contained in neutrophils. MPO has recently been associated with keratinized cells and with decreased postthaw motility in stallion semen [1]. The aim of the study was to determine effects of experimental addition of active MPO on motility, mitochondrial potential, apoptosis induction, membrane and acrosome integrity in equine semen. Semen was collected four times from three stallions. Extended (INRA96Ô) semen was processed for density gradient centrifugation (Equipure Bottom LayerÒ) [2]. Purified pellet was re-extended to 100 x1 06spermatozoa/ ml in INRA96Ô and divided in 3 samples. One sample was used for control, and active human MPO (Calbiochem, Merck) was added in the other two samples to a final concentration of 5 or 50ng/ml. After incubation (2 hours, 20 C), motility was analysed with Computer Assisted Semen Analysis (IVOS, Hamilton-Throne, Beverly, MA, USA) and cytometric analyzes were perfomed with EasyCyte (IMV). Mitochondrial potential and apoptosis were assayed using Guava Mitopotential JC-1 and 7-AAD kit (Millipore). Membrane and acrosome integrity were respectively assayed with PI (Propidium Iodide) (Invitrogen) and PNA (Peanut Agglutinin-Fluorescein Iso Thio Cyanate) (SigmaAldrich). Statistical differences (P < 0.05) were determined using Kruskall-Wallis test. No effect of the stallions was observed on parameters assayed in this study. Unlike total motility, progressive motility was decreased in both MPO concentrations (P < 0.001). MPO addition had no effect on membrane and acrosome integrity. No differences were detected for the percentages of spermatozoa having polarised or depolarised mitochondria. Apoptosis, assayed by 7-AAD fluorescence, was not increased by the treatments. Our results agree with previously published effects on in vitro ROS production systems with xanthine oxidase [3], showing an effect on motility but no influence on mitochondria and membrane or acrosome integrity. However, membrane lipoperoxidation was increased by ROS in this study [3], and it could be linked to the impaired motility also observed in our protocol. Further studies with increasing concentrations of added MPO should be conducted to correlate motility with lipoperoxidation.
509
References [1] Ponthier J, Desvals M, Franck T. de la Rebiere de Pouyade G, Spalart M, Palmer E, Serteyn D, Deleuze S. Myeloperoxidase in equine semen: concentration and localization during freezing processing. J Equine Vet Sci 2012;32:32-7. [2] Edmond AJ, Teague SR, Brinsko SP, Comerford KL, Waite JA, Mancill SS, Love CC, Varner DD. Effect of density-gradient centrifugation on quality and recovery rate of equine spermatozoa. Anim Reprod Sci 2008;107. 318–318. [3] Baumber J, Ball BA, Gravance CG, Medina V, Davies-Morel MC. The effect of reactive oxygen species on equine sperm motility, viability, acrosomal integrity, mitochondrial membrane potential, and membrane lipid peroxidation. J Androl 2000;21:895-902.
Effect of different methods for sperm selection on cooled stallion semen C. Ramires-Neto, G.A. Monteiro, D.J.Z. Delfiol, M.C. Farras, R.R.D. Maziero, F.P. Hartwing, F.O. Papa, and M.A. Alvarenga School of Veterinary Medicine and Animal Science, São Paulo State University, Botucatu, Brazil Sperm selection can improve reproductive efficiency in stallions, primarily in subfertile horses. This sperm selection is made through a centrifugation in density gradient, in which selects morphologically-normal spermatozoa with progressive motility from semen samples. The aim of this work was to assess the effect of sperm selection with different density gradients (PercollÒ and EquipureÒ) on cooled stallion semen. So a preliminary experiment using two ejaculates of 20 stallions was conducted to determine the best force and duration of centrifugation to get the highest sperm quality and recovery rate for each gradient. Then, the ejaculate from 15 stallions was diluted in 1:1 proportion with Botu-SêmenÒ and was divided into three groups as follows: G1, the semen was diluted with BotuSêmenÒ until reaching a concentration of 50 million spermatozoa/mL; G2, the sperm selection was made with EquipureÒ at 300g for 20 minutes; and G3, the sperm selection was made with PercollÒ at 400g for 25 minutes. Pellets from G2 and G3 were diluted with the same extender and concentration as G1. Spermatic motility parameters (CASA-system; HTM-IVOS) and plasma membrane integrity were assessed using epifluorescence microscopy with the fluorescent probes 6-carboxyfluorescein diacetate and iodine propidium in two periods: immediately after semen processing (M0) and after cooled at 15 C for 24 hours (M24). The sperm parameters were compared through analysis of variance (ANOVA) and were considered significant at a probability level of P < 0.05. TM, PM and RAP were lower (P < 0.05) for G1 when compared with G2 and G3 during periods M0 and M24. VAP was different (P < 0.05) between G1 and G2 at M0. No difference regarding HIP was detected between groups (P > 0.05) at any moment, while PMI was increased in both G2 and G3 when compared with G1 at M24. However, G2 had increased sperm recovery percentage (P < 0.05) than G3, 41.7 20.1a and 27.7 16.4b, respectively. In this situation from force and time of centrifugation, EquipureÒ showed better sperm recovery. We conclude that both density gradients (PercollÒ and EquipureÒ) improves quality and
stallion's reproductive tract. In particular, the expression of EQ-12, CD52 and CRISP1 is concentrated in the epididymis. These proteins therefore represent suitable candidates for studies into the mechanisms of protein transfer between the epididymis and spermatozoa during the process of equine sperm maturation.
A flow-cytometric study on the effect of myeloperoxidase on stallion spermatozoal motility and structure J. Ponthier 1, 2, S. Parrila-Hernandez 1, 2, F. Van Den Berghe 1, M. Savic 3, and S. Deleuze 1, 2 1 Equine Clinic, Veterinary Medicine Faculty, Center, ULg University of Liège, B-4000, Liège, Belgium, 2 LINALUXMLS, Centre Européen du Cheval, 18 rue des Champs Elysées, B-5590, Ciney, Belgium, 3 IMV Technologies, ZI n 1 Est, Laigle, F-61300, L'aigle, France Myeloperoxidase (MPO) is a pro-oxidant enzyme that is normally contained in neutrophils. MPO has recently been associated with keratinized cells and with decreased postthaw motility in stallion semen [1]. The aim of the study was to determine effects of experimental addition of active MPO on motility, mitochondrial potential, apoptosis induction, membrane and acrosome integrity in equine semen. Semen was collected four times from three stallions. Extended (INRA96Ô) semen was processed for density gradient centrifugation (Equipure Bottom LayerÒ) [2]. Purified pellet was re-extended to 100 x1 06spermatozoa/ ml in INRA96Ô and divided in 3 samples. One sample was used for control, and active human MPO (Calbiochem, Merck) was added in the other two samples to a final concentration of 5 or 50ng/ml. After incubation (2 hours, 20 C), motility was analysed with Computer Assisted Semen Analysis (IVOS, Hamilton-Throne, Beverly, MA, USA) and cytometric analyzes were perfomed with EasyCyte (IMV). Mitochondrial potential and apoptosis were assayed using Guava Mitopotential JC-1 and 7-AAD kit (Millipore). Membrane and acrosome integrity were respectively assayed with PI (Propidium Iodide) (Invitrogen) and PNA (Peanut Agglutinin-Fluorescein Iso Thio Cyanate) (SigmaAldrich). Statistical differences (P < 0.05) were determined using Kruskall-Wallis test. No effect of the stallions was observed on parameters assayed in this study. Unlike total motility, progressive motility was decreased in both MPO concentrations (P < 0.001). MPO addition had no effect on membrane and acrosome integrity. No differences were detected for the percentages of spermatozoa having polarised or depolarised mitochondria. Apoptosis, assayed by 7-AAD fluorescence, was not increased by the treatments. Our results agree with previously published effects on in vitro ROS production systems with xanthine oxidase [3], showing an effect on motility but no influence on mitochondria and membrane or acrosome integrity. However, membrane lipoperoxidation was increased by ROS in this study [3], and it could be linked to the impaired motility also observed in our protocol. Further studies with increasing concentrations of added MPO should be conducted to correlate motility with lipoperoxidation.
509
References [1] Ponthier J, Desvals M, Franck T. de la Rebiere de Pouyade G, Spalart M, Palmer E, Serteyn D, Deleuze S. Myeloperoxidase in equine semen: concentration and localization during freezing processing. J Equine Vet Sci 2012;32:32-7. [2] Edmond AJ, Teague SR, Brinsko SP, Comerford KL, Waite JA, Mancill SS, Love CC, Varner DD. Effect of density-gradient centrifugation on quality and recovery rate of equine spermatozoa. Anim Reprod Sci 2008;107. 318–318. [3] Baumber J, Ball BA, Gravance CG, Medina V, Davies-Morel MC. The effect of reactive oxygen species on equine sperm motility, viability, acrosomal integrity, mitochondrial membrane potential, and membrane lipid peroxidation. J Androl 2000;21:895-902.
Effect of different methods for sperm selection on cooled stallion semen C. Ramires-Neto, G.A. Monteiro, D.J.Z. Delfiol, M.C. Farras, R.R.D. Maziero, F.P. Hartwing, F.O. Papa, and M.A. Alvarenga School of Veterinary Medicine and Animal Science, São Paulo State University, Botucatu, Brazil Sperm selection can improve reproductive efficiency in stallions, primarily in subfertile horses. This sperm selection is made through a centrifugation in density gradient, in which selects morphologically-normal spermatozoa with progressive motility from semen samples. The aim of this work was to assess the effect of sperm selection with different density gradients (PercollÒ and EquipureÒ) on cooled stallion semen. So a preliminary experiment using two ejaculates of 20 stallions was conducted to determine the best force and duration of centrifugation to get the highest sperm quality and recovery rate for each gradient. Then, the ejaculate from 15 stallions was diluted in 1:1 proportion with Botu-SêmenÒ and was divided into three groups as follows: G1, the semen was diluted with BotuSêmenÒ until reaching a concentration of 50 million spermatozoa/mL; G2, the sperm selection was made with EquipureÒ at 300g for 20 minutes; and G3, the sperm selection was made with PercollÒ at 400g for 25 minutes. Pellets from G2 and G3 were diluted with the same extender and concentration as G1. Spermatic motility parameters (CASA-system; HTM-IVOS) and plasma membrane integrity were assessed using epifluorescence microscopy with the fluorescent probes 6-carboxyfluorescein diacetate and iodine propidium in two periods: immediately after semen processing (M0) and after cooled at 15 C for 24 hours (M24). The sperm parameters were compared through analysis of variance (ANOVA) and were considered significant at a probability level of P < 0.05. TM, PM and RAP were lower (P < 0.05) for G1 when compared with G2 and G3 during periods M0 and M24. VAP was different (P < 0.05) between G1 and G2 at M0. No difference regarding HIP was detected between groups (P > 0.05) at any moment, while PMI was increased in both G2 and G3 when compared with G1 at M24. However, G2 had increased sperm recovery percentage (P < 0.05) than G3, 41.7 20.1a and 27.7 16.4b, respectively. In this situation from force and time of centrifugation, EquipureÒ showed better sperm recovery. We conclude that both density gradients (PercollÒ and EquipureÒ) improves quality and