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Production of reactive oxygen species is lower in stallion spermatozoa after Single Layer Centrifugation with Androcoll-E
6th ISSR Abstracts / Journal of Equine Veterinary Science 32 (2012) 475-518
500
motility (LM) and curvilinear velocity (VCL). The sperm viability was assessed by flow cytometry (SYBR 14/Propidium iodide). The total number of samples was 12; 6 with each freezing extenders. Statistically, there were no significant differences, but nevertheless the following results were observed: samples frozen in Caceres extender showed the best results in membrane integrity post-thaw. Specifically, the mean percentage of live spermatozoa was 31.5 3.8 % for Caceres and 24.8 3.8 % for Botu-crioÒ. Regarding motility and velocity, samples frozen in BotucrioÒ showed slightly higher values compared to the other group. The total motility (TM%) value for the Botu-crioÒ samples was 59.8 5.3% while Caceres-frozen samples showed a value of 55.7 5.3%. This tendency was also observed in the percentage of linearly motile sperm (LM%) 32.0 3.34%, while Caceres frozen samples showed a value of 25.0 3.3%. In the case of circular velocity (VCL), the average velocity for Botu-crioÒ was 59.1 2.07m/s and the VCL in Caceres-frozen samples was 55.1 2.07m/s. In conclusion, these results did not show any difference between the extenders tested. Caceres extender is therefore a suitable option for future freezing protocols.
SLC-samples and uncentrifuged controls were stained with hydroethidine (HE) and dichlorodihydrofluorescein diacetate (DCFDA) to measure ROS production by flow cytometry [2]. Menadione was added to additional aliquots as a control that the spermatozoa were capable of producing ROS [3]. Motility was measured in all samples by computer assisted sperm analysis (SpermVision; Minitube, Tiefenbach, Germany) on arrival at SLU (24h after semen collection) and also at 48h and 72h. Mean values were compared by ANOVA. Results: The proportion of viable non-HE-fluorescent spermatozoa was higher in SLC samples than in controls (54 16% versus 44 17%; P < 0.05) whereas the proportion of viable HE-fluorescent and viable DCFDA-fluorescent spermatozoa was lower in SLC samples than controls (HE:16 12% versus 23 17%, P < 0.05; DCFDA (0.3 0.8% versus 1.0 2.0%; P < 0.05). The proportions of dead HE-fluorescent and dead DCFDA-fluorescent spermatozoa were not different between the two groups. The proportions of ROS-producing spermatozoa increased dramatically after menadione stimulation (P < 0.001) (Table 1). References
Production of reactive oxygen species is lower in stallion spermatozoa after Single Layer Centrifugation with Androcoll-E
[1] Morrell et al, Theriogenology 2009:72, 879–884. [2] van Wienen, et al. ISRN Veterinary Science 2011; doi:10.5402/2011/ 548385 [3] Guthrie & Welch. Journal. Animal Science 2006;84:2089-100.
J.M. Morrell 1, C. Winblad 1, and A. Johannisson 2 1 Clinical Sciences, Swedish University of Agricultural Sciences (SLU), Uppsala, Sweden, 2 Anatomy, Physiology and Biochemistry, Swedish University of Agricultural Sciences (SLU), Uppsala, Sweden
Comparison of seminal parameters in pairs of doses of stallion semen collected at various intervals and their suitability for cryopreservation
High levels of reactive oxygen species (ROS) may be detrimental to stallion sperm survival. Since ROS are produced as byproducts of metabolism, highly metabolising spermatozoa would be expected to produce more ROS than those with a lower metabolic rate. Single Layer Centrifugation (SLC) has been shown to select highly motile spermatozoa which retain their motility for longer than uncentrifuged spermatozoa but their ROS-production has not been measured previously. Objective: to measure ROSproduction in SLC-selected and control (uncentrifuged) stallion spermatozoa. Materials and Methods: cooled semen doses (3-4) from each of 9 stallions were transported to SLU overnight in an insulated box with a cold pack. Aliquots (15 mL) were prepared by SLC [1] and resuspended in INRA96 (IMV Technologies, lí Aigle, France).
M. Mrackova, A. Vinkler, E. Vavrouchov, and M. Sedlinska Equine Clinic, University of Veterinary and Pharmaceutical Sciences Brno, Brno, Czech Republic The objective of this study was to assess the effect of interval of semen collection in one day on seminal parameters and suitability for cryopreservation of stallions ejaculate. Semen samples were obtained from 20 stallions during normal breeding season, 41 pairs of ejaculates were used in this study and they were divided into 4 groups. Group A: double collection up to 90 min (n ¼ 14). Group B: double collection in 90-120 min (n ¼ 12). Group C: double collection in 120-180 min (n ¼ 11). Group D: double collection after more than 180 min (n¼4). Gel-free volume, sperm concentration, motility and total sperm count were measured. After seminal plasma removal, adding extender
Table 1 Change in HE- or DCFDA-fluorescence after menadione stimulation Sample
Treatment
Living HE-
Living HE+
Dead HE+
Living DCFDA-
Living DCFDA+
Dead DCFDA-
Dead DCFDA-
Controls
-men +men -men +men
5618 1314 5417 3625
1218 5618 1614 3623
3010 1911 3012 2711
3211 137 7012 8.614
12 6213 0.30.8 6419
1810 4.55 2911 5.98
1.60.4 28.511 0.31.4 2212
SLC
Progressive motility was higher in SLC samples (62 8%) than controls (56 8) at 24h and remained higher at 48h (53 11% versus 40 13) and 72h (48 14% versus 26 14%). Conclusion: SLC selects motile spermatozoa with a lower proportion of HE-fluorescent and DCFDA-fluorescent cells than uncentrifuged controls, although they are capable of producing ROS when stimulated with menadione. The reduced ROS-production may contribute to enhanced sperm survival during storage.
500
motility (LM) and curvilinear velocity (VCL). The sperm viability was assessed by flow cytometry (SYBR 14/Propidium iodide). The total number of samples was 12; 6 with each freezing extenders. Statistically, there were no significant differences, but nevertheless the following results were observed: samples frozen in Caceres extender showed the best results in membrane integrity post-thaw. Specifically, the mean percentage of live spermatozoa was 31.5 3.8 % for Caceres and 24.8 3.8 % for Botu-crioÒ. Regarding motility and velocity, samples frozen in BotucrioÒ showed slightly higher values compared to the other group. The total motility (TM%) value for the Botu-crioÒ samples was 59.8 5.3% while Caceres-frozen samples showed a value of 55.7 5.3%. This tendency was also observed in the percentage of linearly motile sperm (LM%) 32.0 3.34%, while Caceres frozen samples showed a value of 25.0 3.3%. In the case of circular velocity (VCL), the average velocity for Botu-crioÒ was 59.1 2.07m/s and the VCL in Caceres-frozen samples was 55.1 2.07m/s. In conclusion, these results did not show any difference between the extenders tested. Caceres extender is therefore a suitable option for future freezing protocols.
SLC-samples and uncentrifuged controls were stained with hydroethidine (HE) and dichlorodihydrofluorescein diacetate (DCFDA) to measure ROS production by flow cytometry [2]. Menadione was added to additional aliquots as a control that the spermatozoa were capable of producing ROS [3]. Motility was measured in all samples by computer assisted sperm analysis (SpermVision; Minitube, Tiefenbach, Germany) on arrival at SLU (24h after semen collection) and also at 48h and 72h. Mean values were compared by ANOVA. Results: The proportion of viable non-HE-fluorescent spermatozoa was higher in SLC samples than in controls (54 16% versus 44 17%; P < 0.05) whereas the proportion of viable HE-fluorescent and viable DCFDA-fluorescent spermatozoa was lower in SLC samples than controls (HE:16 12% versus 23 17%, P < 0.05; DCFDA (0.3 0.8% versus 1.0 2.0%; P < 0.05). The proportions of dead HE-fluorescent and dead DCFDA-fluorescent spermatozoa were not different between the two groups. The proportions of ROS-producing spermatozoa increased dramatically after menadione stimulation (P < 0.001) (Table 1). References
Production of reactive oxygen species is lower in stallion spermatozoa after Single Layer Centrifugation with Androcoll-E
[1] Morrell et al, Theriogenology 2009:72, 879–884. [2] van Wienen, et al. ISRN Veterinary Science 2011; doi:10.5402/2011/ 548385 [3] Guthrie & Welch. Journal. Animal Science 2006;84:2089-100.
J.M. Morrell 1, C. Winblad 1, and A. Johannisson 2 1 Clinical Sciences, Swedish University of Agricultural Sciences (SLU), Uppsala, Sweden, 2 Anatomy, Physiology and Biochemistry, Swedish University of Agricultural Sciences (SLU), Uppsala, Sweden
Comparison of seminal parameters in pairs of doses of stallion semen collected at various intervals and their suitability for cryopreservation
High levels of reactive oxygen species (ROS) may be detrimental to stallion sperm survival. Since ROS are produced as byproducts of metabolism, highly metabolising spermatozoa would be expected to produce more ROS than those with a lower metabolic rate. Single Layer Centrifugation (SLC) has been shown to select highly motile spermatozoa which retain their motility for longer than uncentrifuged spermatozoa but their ROS-production has not been measured previously. Objective: to measure ROSproduction in SLC-selected and control (uncentrifuged) stallion spermatozoa. Materials and Methods: cooled semen doses (3-4) from each of 9 stallions were transported to SLU overnight in an insulated box with a cold pack. Aliquots (15 mL) were prepared by SLC [1] and resuspended in INRA96 (IMV Technologies, lí Aigle, France).
M. Mrackova, A. Vinkler, E. Vavrouchov, and M. Sedlinska Equine Clinic, University of Veterinary and Pharmaceutical Sciences Brno, Brno, Czech Republic The objective of this study was to assess the effect of interval of semen collection in one day on seminal parameters and suitability for cryopreservation of stallions ejaculate. Semen samples were obtained from 20 stallions during normal breeding season, 41 pairs of ejaculates were used in this study and they were divided into 4 groups. Group A: double collection up to 90 min (n ¼ 14). Group B: double collection in 90-120 min (n ¼ 12). Group C: double collection in 120-180 min (n ¼ 11). Group D: double collection after more than 180 min (n¼4). Gel-free volume, sperm concentration, motility and total sperm count were measured. After seminal plasma removal, adding extender
Table 1 Change in HE- or DCFDA-fluorescence after menadione stimulation Sample
Treatment
Living HE-
Living HE+
Dead HE+
Living DCFDA-
Living DCFDA+
Dead DCFDA-
Dead DCFDA-
Controls
-men +men -men +men
5618 1314 5417 3625
1218 5618 1614 3623
3010 1911 3012 2711
3211 137 7012 8.614
12 6213 0.30.8 6419
1810 4.55 2911 5.98
1.60.4 28.511 0.31.4 2212
SLC
Progressive motility was higher in SLC samples (62 8%) than controls (56 8) at 24h and remained higher at 48h (53 11% versus 40 13) and 72h (48 14% versus 26 14%). Conclusion: SLC selects motile spermatozoa with a lower proportion of HE-fluorescent and DCFDA-fluorescent cells than uncentrifuged controls, although they are capable of producing ROS when stimulated with menadione. The reduced ROS-production may contribute to enhanced sperm survival during storage.