- Email: [email protected]
Effects of ascorbic acid and pentoxifylline on equine cryopreserved semen submitted to in vitro incubation
Theriogenology 58 (2002) 257±260
Abstract
Effects of ascorbic acid and pentoxifylline on equine cryopreserved semen submitted to in vitro incubation A. Marquesa, R.P. Arrudaa,*, E.C.C. Celeghinia, A.A.O. Gobessob, J.R. Neves Netoc a
Faculdade de Medicina VeterinaÂria, Centro de Biotecnologia em ReproducËaÄo Animal, Universidade de SaÄo Paulo±USP, Pirassununga, SP, Brazil b Departamento de NutricËaÄo e ProducËaÄo Animal, Faculdade de Medicina VeterinaÂria, Universidade de SaÄo Paulo±USP, Pirassununga, SP, Brazil c UNESPÐDepartamento de ReproducËaÄo Animal, Botucatu, SP, Brazil
1. Introduction The use of cryopreserved equine semen is limited by its relatively short-term fertilizing capacity or reduction in the longevity of spermatozoa after thawing. Sperm damaged by freezing and thawing produced signi®cantly more reactive oxygen species (ROS) than live sperm [1]. An important reason for the decrease in motility with consequent decrease in fertility is the peroxidation of sperm membrane lipids in the presence of oxygen radicals [2]. The process of peroxidation induces structural alterations, particularly in the acrosomal region of the sperm cell, a fast and irreversible loss of motility, a deep change in metabolism and a high rate of intracellular components release [3]. The addition of ascorbic acid in chilled-stored stallion semen increased the percentage of membrane intact spermatozoa, when compared with that of the control [2]. Pentoxifylline is a metylxanthine derivative haemorrheologic agent often used in the treatment of peripheral vascular disorders, but when used with semen it acts as a phosphodiesterase inhibitor that can increase intracellular cAMP levels with improvement of motility in cats and humans [4,5]. However, the effects of pentoxifyline on motility of cryopreserved stallion spermatozoa has been little studied [6]. The objective of this study was to evaluate the effects of ascorbic acid, pentoxifylline and the combination of both on the progressive motility, vigor, viability and integrity of acrosome of cryopreserved stallion spermatozoa, submitted to in vitro incubation. * Corresponding author. E-mail address: [email protected] (R.P. Arruda).
0093-691X/02/$ ± see front matter # 2002 Elsevier Science Inc. All rights reserved. PII: S 0 0 9 3 - 6 9 1 X ( 0 2 ) 0 0 8 7 2 - 5
258
A. Marques et al. / Theriogenology 58 (2002) 257±260
2. Materials and methods 2.1. Sperm cryopreservation Four ejaculates were collected from each of ®ve light-horse stallions. Semen was extended in a nonfat, milk-based extender at a concentration of 50 106 cells. Extended semen was then split in 50 ml conical centrifuge tubes and then centrifuged at room temperature at 500g for 15 min to remove seminal plasma. After centrifugation, the supernatant was removed, and the sperm pellet was resuspended to a concentration of 200 106 cells/ml in a cryopreservation media (lactose-egg-yolk extender [7]) with glycerol (5%; v:v). Extended semen was packaged into 0.5 ml straws. It was immediately placed horizontally on a grid 3 cm above liquid nitrogen (10 min.) to be frozen. Straws were then plunged into liquid nitrogen and stored at 196 8C. 2.2. Thawing and dilution of semen Three straws of cryopreserved semen of the same collection and stallion were thawed at 37 8C for 30 s. Samples of semen were diluted following four treatments: (T1) (control), 200 ml of semen 200 ml of TALP; (T2) 200 ml of semen 200 ml of TALP 4:5 g/l of ascorbic acid; (T3) 200 ml of semen200 ml of TALP 3:5 mM of pentoxifyline; and (T4), 200 ml of semen 200 ml of TALP 4:5 g/l of ascorbic acid 3:5 mM of pentoxifyline. All the samples of semen (four treatments) were diluted at ®nal concentration of 50 106 sperm/ ml, placed in 1.5 ml microcentrifuge tubes and maintained in water bath (38 8C) for 120 min. 2.3. Sperm motility and vigor analyses At 0, 60 and 120 min, 8 ml aliquots were placed on pre-warmed slides and the motility (%) and vigor (1±5 score) were analysed on phase contrast microscopy (200). 2.4. Sperm viabillity and acrosomal integrity To assess numbers of live sperm with intact acrosomes (LAI), at time 0, 60, and 120 min, 7 ml of diluted semen (one from each of four treatments) was smeared onto slides. Slides were allowed to dry at room temperature for 2 h, and then stained by the Trypan Blue/ Â CKS et al. [8]. Giemsa method, as described by KOVA The data were analysed by ANOVA. For statistical analyses, percentage values were arcsine transformed The effects of the stallion, treatment and the interactions among them were studied. Individual means were compared by Fisher's protected least signi®cant difference (PLSD), when the main effects were signi®cant (P < 0:05). All analyses were performed using StatView1. 3. Results The means of percentage of progressive motility at time 0 for the control (T1), ascorbic acid (T2), pentoxifylline (T3) and ascorbic acid plus pentoxifylline (T4) treatments were 47.7, 47.5, 52.2 and 52.7%, respectively. The mean of progressive motility in T1 and T2
A. Marques et al. / Theriogenology 58 (2002) 257±260
259
treatments were signi®cantly different (P < 0:05) to the progressive motility in T3 and T4. There was no signi®cant difference (P > 0:05) in progressive motility percentage means at time 60 min for all treatments. At time 120 min the means of progressive motility for the T1, T2, T3 and T4 treatments were 18.7, 16.2, 26.2 and 26.0%, respectively. The T1 and T2 were signi®cantly different (P < 0:05) to the T3 and T4 treatments. The vigor of motility showed statistical differences at time 0, 60, 120 min of incubation. At the ®nal time of 120 min, the means of vigor for the treatments T1, T2, T3 and T4 were 1.67, 1.52, 2.00 and 2.00, respectively. We found statistical differences (P < 0:05) between T1 and T3 and between T1 and T4; however, there was no difference (P > 0:05) between T3 and T4 treatments. There were no statistical differences among the T1, T2, T3 and T4 treatments at time 0, 60 and 120 min for LAI. 4. Discussion In this study, we investigated the effects ascorbic acid, pentoxifylline and the combination of both on the progressive motility, vigor, viability and integrity of the acrosome of cryopreserved stallion spermatozoa, submitted to in vitro incubation. We observed that the progressive motility increased when pentoxifylline was added alone or in combination with ascorbic acid to frozen-thawed stallion semen at time 0 or 120 min of in vitro incubation. Similar results were found by Lewis et al. [9], who reported that pentoxifylline had a highly signi®cant effect on the number of motile and progressively motile human sperm. However, we noted a smaller degree of difference for progressive motility at time 0 than at 120 min between control (T1) and ascorbic acid (T2) treatments when compared with pentoxifylline (T3)andascorbic acidpluspentoxifylline(T4)treatments.Theseobservations suggestthatthe pentoxifylline or the ascorbic acid plus pentoxifylline, as well as improving the progressive motility and sperm vigor, could maintain the better motility and vigor values for a longer time. Thiseffect is desirable, sincethe spermatozoathatmoveprogressively forward are more likely to penetrate the microstructure of the cervix, or successfully cross the uterotubal junction [10]. This study demonstrates that both groups, pentoxifylline and ascorbic acid plus pentoxifylline at time 0 and 120 min of incubation increase semen progressive motility signi®cantly. The ascorbic acid alone did not show signi®catally increased LAI. It may be that in this time period, the production of ROS does not affect the percentage of LAI, disguising the protection by ascorbic acid in the cryopreserved stallion semen. Acknowledgements The authors thank Drs. I.K.M. Liu and B.A. Ball (University of California, Davis, CA) for scienti®c assistance and the FAPESP for ®nancial support. References [1] Ball BA, Gravance CG. Effect of oxidative stress on equine spermatozoa. J Equine Vet Sci 1999;19(9):582. (abstract).
260
A. Marques et al. / Theriogenology 58 (2002) 257±260
[2] Aurich JE, SchoÈnherr U, Hoppe H, Aurich C. Effects of antioxidants on motility and membrane integrity of chilled-stored stallion semen. Theriogenology 1997;48:185±92. [3] Beconi MT, Francia CR, Mora NG, Affranchino MA. Effects of natural antioxidants on frozen bovine semen preservation. Theriogenology 1993;40:841±51. [4] Shen M, Chiang P, Yang R, Chuang H, Chen S. Pentoxifylline stimulates human sperm motility both in vitro and after oral therapy. Br J Clin Pharm 1991;31:711±4. [5] Stachecki JJ, Ginsburg KA, Armant DR. Stimulation of cryopreserved epididymal spermatozoa of the domestic cat using the motility stimulants caffeine, pentoxifylline and 20 -deoxyadenisine. J Androl 1994;15:157±64. [6] Gradill CM, Ball BA. The effects of pentoxifylline to improve motility of cryopreserved equine spermatozoa. Theriogenology 2000;54:1041±7. [7] Martin JC, Klug E, GuÈnsel AR. Centrifugation of stallion semen and its storage in large volume straws. J Reprod Fert Suppl 1979;27:47±51. [8] KovaÂcs A, Foote RH, Nagy SZ, Boersma A, Leidl W, Stolla R, Domes U. Live/dead and acrosome staining of stallion spermatozoa. Int Cong Anim Reprod Stockolm. 2000. p. 82 (abstract) [9] Lewis SEM, Mickinney KA, Thompson W. In¯uence of pentoxifylline on human sperm motility in asthenozoospermic individuals using computer-assisted analysis. Arch Androl 1994;32:175±83. [10] Scott MA. A glimpse at sperm function in vivo: sperm transport and epithelial interaction in the female reproductive in the female reproductive tract. Anim Reprod Sci 2000;60-61:337±48.
Abstract
Effects of ascorbic acid and pentoxifylline on equine cryopreserved semen submitted to in vitro incubation A. Marquesa, R.P. Arrudaa,*, E.C.C. Celeghinia, A.A.O. Gobessob, J.R. Neves Netoc a
Faculdade de Medicina VeterinaÂria, Centro de Biotecnologia em ReproducËaÄo Animal, Universidade de SaÄo Paulo±USP, Pirassununga, SP, Brazil b Departamento de NutricËaÄo e ProducËaÄo Animal, Faculdade de Medicina VeterinaÂria, Universidade de SaÄo Paulo±USP, Pirassununga, SP, Brazil c UNESPÐDepartamento de ReproducËaÄo Animal, Botucatu, SP, Brazil
1. Introduction The use of cryopreserved equine semen is limited by its relatively short-term fertilizing capacity or reduction in the longevity of spermatozoa after thawing. Sperm damaged by freezing and thawing produced signi®cantly more reactive oxygen species (ROS) than live sperm [1]. An important reason for the decrease in motility with consequent decrease in fertility is the peroxidation of sperm membrane lipids in the presence of oxygen radicals [2]. The process of peroxidation induces structural alterations, particularly in the acrosomal region of the sperm cell, a fast and irreversible loss of motility, a deep change in metabolism and a high rate of intracellular components release [3]. The addition of ascorbic acid in chilled-stored stallion semen increased the percentage of membrane intact spermatozoa, when compared with that of the control [2]. Pentoxifylline is a metylxanthine derivative haemorrheologic agent often used in the treatment of peripheral vascular disorders, but when used with semen it acts as a phosphodiesterase inhibitor that can increase intracellular cAMP levels with improvement of motility in cats and humans [4,5]. However, the effects of pentoxifyline on motility of cryopreserved stallion spermatozoa has been little studied [6]. The objective of this study was to evaluate the effects of ascorbic acid, pentoxifylline and the combination of both on the progressive motility, vigor, viability and integrity of acrosome of cryopreserved stallion spermatozoa, submitted to in vitro incubation. * Corresponding author. E-mail address: [email protected] (R.P. Arruda).
0093-691X/02/$ ± see front matter # 2002 Elsevier Science Inc. All rights reserved. PII: S 0 0 9 3 - 6 9 1 X ( 0 2 ) 0 0 8 7 2 - 5
258
A. Marques et al. / Theriogenology 58 (2002) 257±260
2. Materials and methods 2.1. Sperm cryopreservation Four ejaculates were collected from each of ®ve light-horse stallions. Semen was extended in a nonfat, milk-based extender at a concentration of 50 106 cells. Extended semen was then split in 50 ml conical centrifuge tubes and then centrifuged at room temperature at 500g for 15 min to remove seminal plasma. After centrifugation, the supernatant was removed, and the sperm pellet was resuspended to a concentration of 200 106 cells/ml in a cryopreservation media (lactose-egg-yolk extender [7]) with glycerol (5%; v:v). Extended semen was packaged into 0.5 ml straws. It was immediately placed horizontally on a grid 3 cm above liquid nitrogen (10 min.) to be frozen. Straws were then plunged into liquid nitrogen and stored at 196 8C. 2.2. Thawing and dilution of semen Three straws of cryopreserved semen of the same collection and stallion were thawed at 37 8C for 30 s. Samples of semen were diluted following four treatments: (T1) (control), 200 ml of semen 200 ml of TALP; (T2) 200 ml of semen 200 ml of TALP 4:5 g/l of ascorbic acid; (T3) 200 ml of semen200 ml of TALP 3:5 mM of pentoxifyline; and (T4), 200 ml of semen 200 ml of TALP 4:5 g/l of ascorbic acid 3:5 mM of pentoxifyline. All the samples of semen (four treatments) were diluted at ®nal concentration of 50 106 sperm/ ml, placed in 1.5 ml microcentrifuge tubes and maintained in water bath (38 8C) for 120 min. 2.3. Sperm motility and vigor analyses At 0, 60 and 120 min, 8 ml aliquots were placed on pre-warmed slides and the motility (%) and vigor (1±5 score) were analysed on phase contrast microscopy (200). 2.4. Sperm viabillity and acrosomal integrity To assess numbers of live sperm with intact acrosomes (LAI), at time 0, 60, and 120 min, 7 ml of diluted semen (one from each of four treatments) was smeared onto slides. Slides were allowed to dry at room temperature for 2 h, and then stained by the Trypan Blue/ Â CKS et al. [8]. Giemsa method, as described by KOVA The data were analysed by ANOVA. For statistical analyses, percentage values were arcsine transformed The effects of the stallion, treatment and the interactions among them were studied. Individual means were compared by Fisher's protected least signi®cant difference (PLSD), when the main effects were signi®cant (P < 0:05). All analyses were performed using StatView1. 3. Results The means of percentage of progressive motility at time 0 for the control (T1), ascorbic acid (T2), pentoxifylline (T3) and ascorbic acid plus pentoxifylline (T4) treatments were 47.7, 47.5, 52.2 and 52.7%, respectively. The mean of progressive motility in T1 and T2
A. Marques et al. / Theriogenology 58 (2002) 257±260
259
treatments were signi®cantly different (P < 0:05) to the progressive motility in T3 and T4. There was no signi®cant difference (P > 0:05) in progressive motility percentage means at time 60 min for all treatments. At time 120 min the means of progressive motility for the T1, T2, T3 and T4 treatments were 18.7, 16.2, 26.2 and 26.0%, respectively. The T1 and T2 were signi®cantly different (P < 0:05) to the T3 and T4 treatments. The vigor of motility showed statistical differences at time 0, 60, 120 min of incubation. At the ®nal time of 120 min, the means of vigor for the treatments T1, T2, T3 and T4 were 1.67, 1.52, 2.00 and 2.00, respectively. We found statistical differences (P < 0:05) between T1 and T3 and between T1 and T4; however, there was no difference (P > 0:05) between T3 and T4 treatments. There were no statistical differences among the T1, T2, T3 and T4 treatments at time 0, 60 and 120 min for LAI. 4. Discussion In this study, we investigated the effects ascorbic acid, pentoxifylline and the combination of both on the progressive motility, vigor, viability and integrity of the acrosome of cryopreserved stallion spermatozoa, submitted to in vitro incubation. We observed that the progressive motility increased when pentoxifylline was added alone or in combination with ascorbic acid to frozen-thawed stallion semen at time 0 or 120 min of in vitro incubation. Similar results were found by Lewis et al. [9], who reported that pentoxifylline had a highly signi®cant effect on the number of motile and progressively motile human sperm. However, we noted a smaller degree of difference for progressive motility at time 0 than at 120 min between control (T1) and ascorbic acid (T2) treatments when compared with pentoxifylline (T3)andascorbic acidpluspentoxifylline(T4)treatments.Theseobservations suggestthatthe pentoxifylline or the ascorbic acid plus pentoxifylline, as well as improving the progressive motility and sperm vigor, could maintain the better motility and vigor values for a longer time. Thiseffect is desirable, sincethe spermatozoathatmoveprogressively forward are more likely to penetrate the microstructure of the cervix, or successfully cross the uterotubal junction [10]. This study demonstrates that both groups, pentoxifylline and ascorbic acid plus pentoxifylline at time 0 and 120 min of incubation increase semen progressive motility signi®cantly. The ascorbic acid alone did not show signi®catally increased LAI. It may be that in this time period, the production of ROS does not affect the percentage of LAI, disguising the protection by ascorbic acid in the cryopreserved stallion semen. Acknowledgements The authors thank Drs. I.K.M. Liu and B.A. Ball (University of California, Davis, CA) for scienti®c assistance and the FAPESP for ®nancial support. References [1] Ball BA, Gravance CG. Effect of oxidative stress on equine spermatozoa. J Equine Vet Sci 1999;19(9):582. (abstract).
260
A. Marques et al. / Theriogenology 58 (2002) 257±260
[2] Aurich JE, SchoÈnherr U, Hoppe H, Aurich C. Effects of antioxidants on motility and membrane integrity of chilled-stored stallion semen. Theriogenology 1997;48:185±92. [3] Beconi MT, Francia CR, Mora NG, Affranchino MA. Effects of natural antioxidants on frozen bovine semen preservation. Theriogenology 1993;40:841±51. [4] Shen M, Chiang P, Yang R, Chuang H, Chen S. Pentoxifylline stimulates human sperm motility both in vitro and after oral therapy. Br J Clin Pharm 1991;31:711±4. [5] Stachecki JJ, Ginsburg KA, Armant DR. Stimulation of cryopreserved epididymal spermatozoa of the domestic cat using the motility stimulants caffeine, pentoxifylline and 20 -deoxyadenisine. J Androl 1994;15:157±64. [6] Gradill CM, Ball BA. The effects of pentoxifylline to improve motility of cryopreserved equine spermatozoa. Theriogenology 2000;54:1041±7. [7] Martin JC, Klug E, GuÈnsel AR. Centrifugation of stallion semen and its storage in large volume straws. J Reprod Fert Suppl 1979;27:47±51. [8] KovaÂcs A, Foote RH, Nagy SZ, Boersma A, Leidl W, Stolla R, Domes U. Live/dead and acrosome staining of stallion spermatozoa. Int Cong Anim Reprod Stockolm. 2000. p. 82 (abstract) [9] Lewis SEM, Mickinney KA, Thompson W. In¯uence of pentoxifylline on human sperm motility in asthenozoospermic individuals using computer-assisted analysis. Arch Androl 1994;32:175±83. [10] Scott MA. A glimpse at sperm function in vivo: sperm transport and epithelial interaction in the female reproductive in the female reproductive tract. Anim Reprod Sci 2000;60-61:337±48.