- Email: [email protected]
Cryopreservation of epididymal semen: a retrospective study of 101 clinical cases
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Abstracts / Journal of Equine Veterinary Science 43 (2016) S56eS82
lacking. The objective of this study was to compare CC and the use of SF as methods for seminal plasma removal. Variables analysed were sperm recovery rates, sperm morphology, and longevity of cooled-stored semen. Fifteen light-breed stallions (ages 5-22yrs) had semen collected every other day for three ejaculates (n¼45 ejaculates). Ejaculates were collected in a Missouri AV, extended (1:1 v/v) in skim milk based extender, Botu-semen (BS) (BotuPharma, Botucatu, Brazil), and then split into one of three groups: control (CT), SF and CC. Semen in the CT group was further extended to 25 million sperm/mL. For group SF, extended semen (24 mL) was filtered with the SpermFilter (Botu-Pharma, Botucatu, Brazil) until a volume of 7.5mL of concentrated semen remained; the remaining semen was then extended in BS to a concentration of 25 million sperm/mL. Group CC had 24 mL of extended semen centrifuged at 1000g for 20 min in 50 mL conical tubes with 1mL of Cushion Fluid (Minitube). Thereafter, supernatant and cushion fluid were discarded, and the remaining 7.5 mL was re-extended in BS to 25 million/mL. Recovery rates were calculated from concentrations determined by a NucleocounterSP100. Aliquots from all groups were preserved in buffered formol saline, and morphologic analysis was performed with DIC microscopy to assess the percentage of normal sperm per 200 cells. Three 5mL aliquots from each group were packaged (Whirl-Pak®, Nasco), and maintained at 5 C for 72h. Percent total motility (TM) and progressive sperm motility (PM) were determined using a computer assisted sperm analyser immediately after packaging 0h, and 24h, 48h and 72h after semen processing. Motility parameters were analysed by ANOVA, repeated measures and when significant by Tukey’s test. Mann-Whitney Rank Sum test was used to compare percent of normal sperm cells and those with detached heads. Significance was set at p<0.05. Mean recovery rate was significantly higher for CC (100.5 ± 0.3) than for SF (93.0 ± 2.8%) (p<0.05). Overall, the mean TM was higher for all timepoints evaluated for the CC group (24 h: 61±3.9, 48 h: 59 ± 4.1,72 h: 56.7 ± 4.3) compared to the SF group (24 h: 58 ± 4.2, 48 h: 55 ± 4, 72 h 51.6 ± 3.8) (p<0.0001). No significant difference was found between the mean TM for all time points between the CT and CC group, (p>0.3) or the CT and SF group (p¼0.1). However, CC had greater PM sperm compared to the CT and SF groups (p<0.001), post hoc comparisons demonstrated that CC had superior PM at 48 h (51 ± 4) and 72 h (48.1 ± 4.1) after processing (p<0.05). There was no significant difference between the CT and SP groups for PM (p¼0.18) for all time points. There were no differences in the percentage of normal sperm cells and detached heads between the three groups (p>0.05). Whilst this study provides evidence that CC resulted in higher recovery rates and PM sperm, the SF may useful in situations where a centrifuge is unavailable as the differences might be clinically negligible.
was collected from 10 stallions with proven fertility of cooled-stored semen (two ejaculates per stallion) and processed for cooled-storage at 5 C with EquiPlus extender (Minitube, Tiefenbach, Germany). Two final concentrations, 50x106 and 100x106 cells/mL, were used. Semen was analyzed for percent of total motility, percent of progressive motility and membrane integrity (Spermvision, Minitube) immediately after processing and at 24hintervals until 72h of storage. DNA cytosine methylation was assessed by ELISA (5-mC DNA ELISA Kit, Zymo Research, Irvine, CA, USA) after DNA extraction and denaturation (100ng DNA per sample). The level of 5-mC in DNA is reported as the percentage of methylated cytosine relative to the genomic cytosine content. For statistical analysis the General Linear Model (GLM) for repeated measures with semen concentration (50x106 and 100x106 cells/ mL) as within subject factor was used (SPSS Statistics 21). Total motility, progressive motility and membrane integrity decreased over time, but no differences were noted between semen concentrations (e.g. total motility 0h 50x106: 91 ± 0.9%, 100 x 106: 90.2 ± 1%; 24h 50x106: 90.4 ± 1.1%,100x106: 86.9 ± 2.5%, time: p<0.001). Percent of DNA methylation did not change over time and was not affected by semen concentration (0h 50x106: 0.6±0.1%, 100x106: 1.0±0.4%, 24h 50x106: 0.4 ± 0.1%, 100x106: 1.0±0.5%). Among total motility, progressive motility and membrane integrity high correlations were found (e.g. total to progressive motility: r¼0.866, p<0.001, to membrane integrity: r¼0.813, p<0.001) while DNAmethylation was only poorly correlated with total motility (r¼0.225, p<0.01), progressive motility (r¼0.376, p<0.001) and membrane integrity (r¼0.269, p<0.01). The results demonstrate that cooled-storage of equine semen induces no relevant changes in sperm DNA cytosine methylation. If semen concentration was adjusted to 100x106 sperm/mL which is often considered detrimental to quality of especially non-centrifuged cooled-stored stallion semen neither classical parameters motility and membrane integrity nor DNA methylation of sperm DNA were affected under the conditions of the present study.
Key Words: Centrifugation, cooled semen, morphology, seminal plasma, sperm-filter, stallion
Spermatozoa can be recovered from stallion’s epididymides in the event of untimely death, life-threatening terminal medical conditions or as an elective procedure and cryopreserved for future use. The goal of this retrospective study was to report results of epididymal semen harvesting and cryopreservation from stallions in a clinical equine reproduction program. Epididymal semen was harvested from 101 stallions (~20% castration elective/80% injury or illness) with an average age of 13.3 ± 7.3 years (mean ± SD) over a 13 year period. The most commonly reported reasons for epididymal sperm recovery were musculoskeletal trauma and colic. The epididymides were flushed (by retrograde flush) with at least 3 mls of donor seminal plasma (collected from CSU stallions by centrifugation) followed by 10 to 20 ml of air. Freezing extender was added to the recovered semen sample and the sperm concentration subsequently determined with a NucleoCounter® and sperm motility evaluated using a CASA unit (SpermVision®). An
29 Cooled-storage of equine semen does not induce major changes in sperm DNA methylation R.A. Oliveira, N. Ille, D. Scarlet, C. Aurich Vetmeduni Vienna, Vienna, Austria A decrease in the fertility of equine semen during cooled-storage may be caused by changes in sperm membrane function. We have recently shown that cryopreservation of equine semen increases sperm DNA methylation. We therefore hypothesized that semen processing and cooled-storage may also contribute to epigenetic changes in sperm DNA methylation level. For this purpose, semen
Acknowledgements Funds provided by CAPES, Brasília, Brazil. Key Words: DNA methylation, horse, semen storage
30 Cryopreservation of epididymal semen: a retrospective study of 101 clinical cases G.M. Fors, P.D. Moffett, J.K. Graham, P.M. McCue Colorado State University, Fort Collins, CO USA
Abstracts / Journal of Equine Veterinary Science 43 (2016) S56eS82
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average of 22.5 ± 23.5 billion sperm was harvested per stallion. Sperm recovery was too low to cryopreserve in 10 cases. Cryopreservation of sperm was performed on the other 91 cases. In most cases, sperm were cryopreserved using two or more cryoprotectants. As with ejaculated semen, epididymal semen from individual stallions varied considerably among different freezing extenders. In general, a diluent with more than one type of cryoprotectant (i.e. CryoMaxTM or Botu-Crio®) yielded better postthaw epididymal sperm motility than a diluent containing only one cryoprotectant (i.e. only glycerol). Initial total and progressive sperm motility prior to cryopreservation were 48.2 ± 25.0 and 35.2 ± 21.2, respectively. Post-thaw motility for spermatozoa cryopreserved in commercial semen extenders ranged from 38.6 ± 23.1 % total motility and 30.1 ± 21.2 % progressive motility to 16.2 ± 11.3 % total motility and 8.6 ± 6.8 % progressive motility. An average of 24.9 ± 21.5 breeding doses were cryopreserved per stallion. Stallions were categorized into very good ( 50 % postthaw total sperm motility), good (30 to 49 %), fair (15 to 29 %) and poor (< 15 %) freezers. The percentages of stallions in each category were 11.4 %, 24.1 %, 27.8 % and 36.7 %, respectively. In conclusion, harvesting and cryopreservation of epididymal semen was most commonly performed as an emergency procedure associated with a life-threatening medical condition. It is recommended that epididymal semen be subdivided and cryopreserved in more than one freezing extender and that at least one extender containing a combination of cryoprotectants be used in order to optimize post-thaw epididymal sperm motility.
Freezin® MFR5, as recommended by the manufacturer. After thawing, total sperm motility was assessed at 0 and 10 min using CASA; and sperm viability was assessed using flow cytometry. The percentages of motile sperm at 0 min were not different in any commercial extender (46-58%; p>0.05), nor were the percentages of viable sperm measured by flow cytometry (32-37%; p>0.05). However, 10 minutes after thawing the percentages of motile sperm frozen in the different diluents were 50ab, 57a, 52ab, 40b, 36b, respectively; with sperm frozen in diluents containing a combination of cryoprotectants being higher than diluents containing glycerol only (p<0.05). In conclusion, the commercial freezing extenders containing a combination of cryoprotectants tended to preserve stallion sperm motility better than commercial extenders containing only glycerol, as the cryoprotectant.
Key Words: stallion, epididymis, cryopreservation
All metabolizing cells produce reactive oxygen species (ROS) under physiological conditions, including sperm; however some of these are known to have a negative effect on sperm in excessive levels causing DNA damage. Mitochondria are the major site of ROS formation which results in a disruption of electron transport. The importance of these organelles as a source of oxidative stress in semen is an important area for investigation. Therefore, a flow cytometry protocol assay was developed to measure simultaneously mitochondrial generation of O-2 (MSR; MitoSox Red, Life Technologies), high mitochondrial potential (MST; MitoStatus Red, BD Pharmigen) and membrane integrity (SG; SytoxGreen, Life Technologies), associated with Hoechst 33342 (H342; Sigma) for identification of cellular events. Six ejaculates from 3 different stallions were used. Each ejaculate was split into two aliquots. The first sample was maintained (fresh semen) and the other was submitted to flash frozen (liquid nitrogen -196oC) and thawed (water bath (37oC) multiple cycles (6). From these, a third sample was prepared with 1:1 (v:v) proportion of fresh semen: flash frozen. Three treatments were analyzed with the following ratios (dead/alive cells): 0:100 (T0), 50:50 (T50), and 100:0 (T100). For analysis, samples were diluted in TALP sperm medium to a final concentration of 2x106 spermatozoa/mL and in 500mL for each group was added 2mM of MSR, 20nM of MitoStatus Red, 50nM of SG and 7mM of H342 and incubated for 15min at 37 C. Flow cytometry analysis samples were run on a BD LSRFortessa flow cytometer (Becton Dickinson) equipped with 488nm blue laser, one 640nm red laser and 405nm violet laser. For this study, excitation was performed at 488nm and the emission filters used were 695/40nm (MST), 530/30nm (SG), for excitation in 640nm, the emission filter was 660/20nm (MSR) and for excitation in 405nm the emission filter was 450/50nm (H342). At least 10.000 cells per sample were analyzed and data were stored in list mode files. Statistical analysis was performed using GraphPad Prism 5 (2007) linear regression was carried out to determine the relationship between treatments and percentage of fluorescence positive cells. Linear regression indicated a high determination coefficient MSR (r2¼0.95), and MST (r2¼0.92) and SG (r2¼0.94). The developed protocol was sensitive to detection simultaneously
31 Cryopreservation of stallion sperm in freezing diluents containing glycerol alone or a combination of cryoprotectants P. Moffett, G. Fors, J.K. Graham Department of Biomedical Sciences, Colorado State University, Fort Collins, CO, USA Glycerol is the standard cryoprotectant used for cryopreserving stallion sperm. Glycerol, however, has low membrane permeability compared to water and therefore, causes significant osmotic damage to stallion sperm. Alternative cryoprotectants, methyl formamide (MF) and di-methyl formamide have lower molecular weights and are more permeable to the stallion sperm plasma membrane than glycerol, and may, therefore, induce less osmotic damage to the sperm during cryopreservation. Experiments were conducted to determine if a combination of cryoprotectants could protect stallion sperm from cryodamage more effectively than glycerol alone. In Experiment 1, semen (2 ejaculates from each of 5 stallions) was cryopreserved in Lactose-EDTA extender containing either glycerol alone (5%; control) or 1, 2 or 3% glycerol each in combination with 1, 2 or 3% MF. Data were analyzed by ANOVA and treatments means separated using SNK multiple range test. The percentages of total motile sperm for the three MF concentrations within each glycerol level were not different (P>0.05) and for simplicity are averaged together. Postthaw total motility for sperm frozen in 5% glycerol alone (27%) and cryoprotectant combinations containing 1% (27%) and 3% (22%) glycerol were lower than sperm frozen in 2% glycerol in combination with MF (40%; p<0.05). Experiment II, compared the postthaw motility of sperm (3 ejaculates from 5 stallions) frozen in 3 commercially available freezing extenders containing a combination of cryoprotectants: 1) BotuCrio®, 2) E-Z Freezin® Cryomax™ LE, and 3) E-Z Freezin® Cryomax™ MFR5; and 2 freezing extenders containing only glycerol: 4) E-Z Freezin® LE; or 5) E-Z
Key Words: cryopreservation, cryoprotectant mixture, cryodiluents
32 Determination of intracellular and mitochondrial superoxide generation and high mitochondrial membrane potential in equine sperm using flow cytometry C.P. Freitas-Dell’Aqua, Y.F.R. Sancler-Silva, E.R. Silva-Jr, J.A. Dell’Aqua, Jr., F.O. Papa ~o Paulo State University, Botucatu, Sa ~o Paulo, Brazil Sa
Abstracts / Journal of Equine Veterinary Science 43 (2016) S56eS82
lacking. The objective of this study was to compare CC and the use of SF as methods for seminal plasma removal. Variables analysed were sperm recovery rates, sperm morphology, and longevity of cooled-stored semen. Fifteen light-breed stallions (ages 5-22yrs) had semen collected every other day for three ejaculates (n¼45 ejaculates). Ejaculates were collected in a Missouri AV, extended (1:1 v/v) in skim milk based extender, Botu-semen (BS) (BotuPharma, Botucatu, Brazil), and then split into one of three groups: control (CT), SF and CC. Semen in the CT group was further extended to 25 million sperm/mL. For group SF, extended semen (24 mL) was filtered with the SpermFilter (Botu-Pharma, Botucatu, Brazil) until a volume of 7.5mL of concentrated semen remained; the remaining semen was then extended in BS to a concentration of 25 million sperm/mL. Group CC had 24 mL of extended semen centrifuged at 1000g for 20 min in 50 mL conical tubes with 1mL of Cushion Fluid (Minitube). Thereafter, supernatant and cushion fluid were discarded, and the remaining 7.5 mL was re-extended in BS to 25 million/mL. Recovery rates were calculated from concentrations determined by a NucleocounterSP100. Aliquots from all groups were preserved in buffered formol saline, and morphologic analysis was performed with DIC microscopy to assess the percentage of normal sperm per 200 cells. Three 5mL aliquots from each group were packaged (Whirl-Pak®, Nasco), and maintained at 5 C for 72h. Percent total motility (TM) and progressive sperm motility (PM) were determined using a computer assisted sperm analyser immediately after packaging 0h, and 24h, 48h and 72h after semen processing. Motility parameters were analysed by ANOVA, repeated measures and when significant by Tukey’s test. Mann-Whitney Rank Sum test was used to compare percent of normal sperm cells and those with detached heads. Significance was set at p<0.05. Mean recovery rate was significantly higher for CC (100.5 ± 0.3) than for SF (93.0 ± 2.8%) (p<0.05). Overall, the mean TM was higher for all timepoints evaluated for the CC group (24 h: 61±3.9, 48 h: 59 ± 4.1,72 h: 56.7 ± 4.3) compared to the SF group (24 h: 58 ± 4.2, 48 h: 55 ± 4, 72 h 51.6 ± 3.8) (p<0.0001). No significant difference was found between the mean TM for all time points between the CT and CC group, (p>0.3) or the CT and SF group (p¼0.1). However, CC had greater PM sperm compared to the CT and SF groups (p<0.001), post hoc comparisons demonstrated that CC had superior PM at 48 h (51 ± 4) and 72 h (48.1 ± 4.1) after processing (p<0.05). There was no significant difference between the CT and SP groups for PM (p¼0.18) for all time points. There were no differences in the percentage of normal sperm cells and detached heads between the three groups (p>0.05). Whilst this study provides evidence that CC resulted in higher recovery rates and PM sperm, the SF may useful in situations where a centrifuge is unavailable as the differences might be clinically negligible.
was collected from 10 stallions with proven fertility of cooled-stored semen (two ejaculates per stallion) and processed for cooled-storage at 5 C with EquiPlus extender (Minitube, Tiefenbach, Germany). Two final concentrations, 50x106 and 100x106 cells/mL, were used. Semen was analyzed for percent of total motility, percent of progressive motility and membrane integrity (Spermvision, Minitube) immediately after processing and at 24hintervals until 72h of storage. DNA cytosine methylation was assessed by ELISA (5-mC DNA ELISA Kit, Zymo Research, Irvine, CA, USA) after DNA extraction and denaturation (100ng DNA per sample). The level of 5-mC in DNA is reported as the percentage of methylated cytosine relative to the genomic cytosine content. For statistical analysis the General Linear Model (GLM) for repeated measures with semen concentration (50x106 and 100x106 cells/ mL) as within subject factor was used (SPSS Statistics 21). Total motility, progressive motility and membrane integrity decreased over time, but no differences were noted between semen concentrations (e.g. total motility 0h 50x106: 91 ± 0.9%, 100 x 106: 90.2 ± 1%; 24h 50x106: 90.4 ± 1.1%,100x106: 86.9 ± 2.5%, time: p<0.001). Percent of DNA methylation did not change over time and was not affected by semen concentration (0h 50x106: 0.6±0.1%, 100x106: 1.0±0.4%, 24h 50x106: 0.4 ± 0.1%, 100x106: 1.0±0.5%). Among total motility, progressive motility and membrane integrity high correlations were found (e.g. total to progressive motility: r¼0.866, p<0.001, to membrane integrity: r¼0.813, p<0.001) while DNAmethylation was only poorly correlated with total motility (r¼0.225, p<0.01), progressive motility (r¼0.376, p<0.001) and membrane integrity (r¼0.269, p<0.01). The results demonstrate that cooled-storage of equine semen induces no relevant changes in sperm DNA cytosine methylation. If semen concentration was adjusted to 100x106 sperm/mL which is often considered detrimental to quality of especially non-centrifuged cooled-stored stallion semen neither classical parameters motility and membrane integrity nor DNA methylation of sperm DNA were affected under the conditions of the present study.
Key Words: Centrifugation, cooled semen, morphology, seminal plasma, sperm-filter, stallion
Spermatozoa can be recovered from stallion’s epididymides in the event of untimely death, life-threatening terminal medical conditions or as an elective procedure and cryopreserved for future use. The goal of this retrospective study was to report results of epididymal semen harvesting and cryopreservation from stallions in a clinical equine reproduction program. Epididymal semen was harvested from 101 stallions (~20% castration elective/80% injury or illness) with an average age of 13.3 ± 7.3 years (mean ± SD) over a 13 year period. The most commonly reported reasons for epididymal sperm recovery were musculoskeletal trauma and colic. The epididymides were flushed (by retrograde flush) with at least 3 mls of donor seminal plasma (collected from CSU stallions by centrifugation) followed by 10 to 20 ml of air. Freezing extender was added to the recovered semen sample and the sperm concentration subsequently determined with a NucleoCounter® and sperm motility evaluated using a CASA unit (SpermVision®). An
29 Cooled-storage of equine semen does not induce major changes in sperm DNA methylation R.A. Oliveira, N. Ille, D. Scarlet, C. Aurich Vetmeduni Vienna, Vienna, Austria A decrease in the fertility of equine semen during cooled-storage may be caused by changes in sperm membrane function. We have recently shown that cryopreservation of equine semen increases sperm DNA methylation. We therefore hypothesized that semen processing and cooled-storage may also contribute to epigenetic changes in sperm DNA methylation level. For this purpose, semen
Acknowledgements Funds provided by CAPES, Brasília, Brazil. Key Words: DNA methylation, horse, semen storage
30 Cryopreservation of epididymal semen: a retrospective study of 101 clinical cases G.M. Fors, P.D. Moffett, J.K. Graham, P.M. McCue Colorado State University, Fort Collins, CO USA
Abstracts / Journal of Equine Veterinary Science 43 (2016) S56eS82
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average of 22.5 ± 23.5 billion sperm was harvested per stallion. Sperm recovery was too low to cryopreserve in 10 cases. Cryopreservation of sperm was performed on the other 91 cases. In most cases, sperm were cryopreserved using two or more cryoprotectants. As with ejaculated semen, epididymal semen from individual stallions varied considerably among different freezing extenders. In general, a diluent with more than one type of cryoprotectant (i.e. CryoMaxTM or Botu-Crio®) yielded better postthaw epididymal sperm motility than a diluent containing only one cryoprotectant (i.e. only glycerol). Initial total and progressive sperm motility prior to cryopreservation were 48.2 ± 25.0 and 35.2 ± 21.2, respectively. Post-thaw motility for spermatozoa cryopreserved in commercial semen extenders ranged from 38.6 ± 23.1 % total motility and 30.1 ± 21.2 % progressive motility to 16.2 ± 11.3 % total motility and 8.6 ± 6.8 % progressive motility. An average of 24.9 ± 21.5 breeding doses were cryopreserved per stallion. Stallions were categorized into very good ( 50 % postthaw total sperm motility), good (30 to 49 %), fair (15 to 29 %) and poor (< 15 %) freezers. The percentages of stallions in each category were 11.4 %, 24.1 %, 27.8 % and 36.7 %, respectively. In conclusion, harvesting and cryopreservation of epididymal semen was most commonly performed as an emergency procedure associated with a life-threatening medical condition. It is recommended that epididymal semen be subdivided and cryopreserved in more than one freezing extender and that at least one extender containing a combination of cryoprotectants be used in order to optimize post-thaw epididymal sperm motility.
Freezin® MFR5, as recommended by the manufacturer. After thawing, total sperm motility was assessed at 0 and 10 min using CASA; and sperm viability was assessed using flow cytometry. The percentages of motile sperm at 0 min were not different in any commercial extender (46-58%; p>0.05), nor were the percentages of viable sperm measured by flow cytometry (32-37%; p>0.05). However, 10 minutes after thawing the percentages of motile sperm frozen in the different diluents were 50ab, 57a, 52ab, 40b, 36b, respectively; with sperm frozen in diluents containing a combination of cryoprotectants being higher than diluents containing glycerol only (p<0.05). In conclusion, the commercial freezing extenders containing a combination of cryoprotectants tended to preserve stallion sperm motility better than commercial extenders containing only glycerol, as the cryoprotectant.
Key Words: stallion, epididymis, cryopreservation
All metabolizing cells produce reactive oxygen species (ROS) under physiological conditions, including sperm; however some of these are known to have a negative effect on sperm in excessive levels causing DNA damage. Mitochondria are the major site of ROS formation which results in a disruption of electron transport. The importance of these organelles as a source of oxidative stress in semen is an important area for investigation. Therefore, a flow cytometry protocol assay was developed to measure simultaneously mitochondrial generation of O-2 (MSR; MitoSox Red, Life Technologies), high mitochondrial potential (MST; MitoStatus Red, BD Pharmigen) and membrane integrity (SG; SytoxGreen, Life Technologies), associated with Hoechst 33342 (H342; Sigma) for identification of cellular events. Six ejaculates from 3 different stallions were used. Each ejaculate was split into two aliquots. The first sample was maintained (fresh semen) and the other was submitted to flash frozen (liquid nitrogen -196oC) and thawed (water bath (37oC) multiple cycles (6). From these, a third sample was prepared with 1:1 (v:v) proportion of fresh semen: flash frozen. Three treatments were analyzed with the following ratios (dead/alive cells): 0:100 (T0), 50:50 (T50), and 100:0 (T100). For analysis, samples were diluted in TALP sperm medium to a final concentration of 2x106 spermatozoa/mL and in 500mL for each group was added 2mM of MSR, 20nM of MitoStatus Red, 50nM of SG and 7mM of H342 and incubated for 15min at 37 C. Flow cytometry analysis samples were run on a BD LSRFortessa flow cytometer (Becton Dickinson) equipped with 488nm blue laser, one 640nm red laser and 405nm violet laser. For this study, excitation was performed at 488nm and the emission filters used were 695/40nm (MST), 530/30nm (SG), for excitation in 640nm, the emission filter was 660/20nm (MSR) and for excitation in 405nm the emission filter was 450/50nm (H342). At least 10.000 cells per sample were analyzed and data were stored in list mode files. Statistical analysis was performed using GraphPad Prism 5 (2007) linear regression was carried out to determine the relationship between treatments and percentage of fluorescence positive cells. Linear regression indicated a high determination coefficient MSR (r2¼0.95), and MST (r2¼0.92) and SG (r2¼0.94). The developed protocol was sensitive to detection simultaneously
31 Cryopreservation of stallion sperm in freezing diluents containing glycerol alone or a combination of cryoprotectants P. Moffett, G. Fors, J.K. Graham Department of Biomedical Sciences, Colorado State University, Fort Collins, CO, USA Glycerol is the standard cryoprotectant used for cryopreserving stallion sperm. Glycerol, however, has low membrane permeability compared to water and therefore, causes significant osmotic damage to stallion sperm. Alternative cryoprotectants, methyl formamide (MF) and di-methyl formamide have lower molecular weights and are more permeable to the stallion sperm plasma membrane than glycerol, and may, therefore, induce less osmotic damage to the sperm during cryopreservation. Experiments were conducted to determine if a combination of cryoprotectants could protect stallion sperm from cryodamage more effectively than glycerol alone. In Experiment 1, semen (2 ejaculates from each of 5 stallions) was cryopreserved in Lactose-EDTA extender containing either glycerol alone (5%; control) or 1, 2 or 3% glycerol each in combination with 1, 2 or 3% MF. Data were analyzed by ANOVA and treatments means separated using SNK multiple range test. The percentages of total motile sperm for the three MF concentrations within each glycerol level were not different (P>0.05) and for simplicity are averaged together. Postthaw total motility for sperm frozen in 5% glycerol alone (27%) and cryoprotectant combinations containing 1% (27%) and 3% (22%) glycerol were lower than sperm frozen in 2% glycerol in combination with MF (40%; p<0.05). Experiment II, compared the postthaw motility of sperm (3 ejaculates from 5 stallions) frozen in 3 commercially available freezing extenders containing a combination of cryoprotectants: 1) BotuCrio®, 2) E-Z Freezin® Cryomax™ LE, and 3) E-Z Freezin® Cryomax™ MFR5; and 2 freezing extenders containing only glycerol: 4) E-Z Freezin® LE; or 5) E-Z
Key Words: cryopreservation, cryoprotectant mixture, cryodiluents
32 Determination of intracellular and mitochondrial superoxide generation and high mitochondrial membrane potential in equine sperm using flow cytometry C.P. Freitas-Dell’Aqua, Y.F.R. Sancler-Silva, E.R. Silva-Jr, J.A. Dell’Aqua, Jr., F.O. Papa ~o Paulo State University, Botucatu, Sa ~o Paulo, Brazil Sa