Influence of addition of motility stimulators in cryopreservation of sperm from subfertile stallions
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Influence of addition of motility stimulators in cryopreservation of sperm from subfertile stallions C. Ramires-Neto * , G.A. Monteiro, Y.F.R. Sancler-Silva, R.R.D. Maziero, F.P. Lisboa, C.P. Freitas-Dell’Aqua, M.A. Alvarenga, F.O. Papa Department of Animal Reproduction and Veterinary Radiology, School of Veterinary Medicine and Animal Science, UNESP, Botucatu, SP, Brazil Pasquini et al. (Anim Reprod Sci 107:338-339, 2008) observed that adding sperm motility stimulants improves motility of epididymal sperm after freezing and thawing. Morris et al. (Theriogenology 58:643-646, 2002) found an improvement in fertility when cryopreserved epididymal sperm were added to Sperm-TALP prior to freezing. The aim of the present study was to evaluate the effect of different motility stimulants on cryopreserved semen from sub- fertile stallions. Six stallions of different breeds with a history of low fertility (pregnancy rate < 25%) were used. Semen was collected using an artificial vagina, and semen dilution (1:1) was performed with Botu-Semen TM (Botu- pharma, Brazil). After dilution, 20% Botu-Semen TM (group CG), Sperm-TALP (group GS, Human Tubal Fluid medium supplemented with 0.5mg/mL caffeine, 100IU/mL penicillin and 100g/mL streptomycin) or Fert-TALP (group GF, Human Tubal Fluid medium supplemented with 0.5mg/mL caffeine, 0.003 g/mL heparin, 0.0003g/mL penicillamine, 0.00011g/mL hypotaurine, 0.00018g/mL epinephrine, 100 IU/mL penicillin and 100g/mL streptomycin) was added to the extended semen. Samples were incubated for 15 min at 37 C and then centrifuged at 600xg for 10 min. The su- pernatant was discarded, and the pellet was resuspended with Botu-Cryo TM at a concentration of 100x10 6 total sperm/mL. The semen was packaged in 0.5-mL straws and cryopreserved (sequentially: 5 C/20 min; -120oC/20 min, followed by immersion in liquid nitrogen). The straws were thawed for 20 sec at 46 C. After incubation at 37 C (M1) and after freezing and thawing (M2) the sperm kinetic analysis was performed by CASA (HTM-IVOS, IMV, USA). At M2, plasma membrane integrity (MI, probes propidium iodide and FITC-PSA), translocation of membrane phos- pholipids (TP, annexin V) and DNA fragmentation assess- ment (DNA, probe Acridine Orange) were determined by flow cytometry (BD LSR Fortessa, BD, Mountain View, USA). All sperm parameters were compared by analysis of vari- ance (ANOVA). The significance level considered was 5%. No difference was observed (p>0.05) at M1 in total motility (TM%, GC¼24.1 16.1, GS¼26.315.8, GF¼29.317.8), pro- gressive motility (PM%, GC¼8.58.5, GS¼8.37.6, GF¼9.59.0), progressive angular velocity (VAP, mm/s, GC¼101.314.3, GS¼106.611.8, GF¼74.844.4) and percentage of rapid sperm (RAP%; GC¼17.512.8, GS¼ 19.613.4, GF¼22.1 15.3). At M2 there were no differences (p>0.05) in TM (GC¼16.215.0, GS¼19.817.4, GF¼16.3 14.7), PM (GC¼7.58.5, GS¼10.1 10.5, GF¼7.89.7), VAP (GC¼75.822.8, GS¼81.025.2, GF¼78.818.1), RAP (GC¼10.1 11.3, GS¼14.014.0, GF¼10.1 11.1), MI (%, GC¼ 15.14.8, GS¼15.26.4, GF¼14.57.9), TP (%, GC¼6.910.7, GS¼4.54.5, GF¼3.53.2) and DNA (GC¼2.60.6, GS¼2.7 0.6, GF¼2.50.6) among the groups. In conclusion, there was no effect of addition of motility stimulators on sperm kinetic parameters and flow cytometric parameters for frozen semen from subfertile stallions. Acknowledgments FAPESP. * Presenting author Contents lists available at ScienceDirect Journal of Equine Veterinary Science journal homepage: www.j-evs.com Journal of Equine Veterinary Science 34 (2014) 98
Transcript of Influence of addition of motility stimulators in cryopreservation of sperm from subfertile stallions
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Journal of Equine Veterinary Science 34 (2014) 98
Contents lists ava
Journal of Equine Veterinary Science
journal homepage: www.j -evs.com
Influence of addition of motility stimulators incryopreservation of sperm from subfertile stallions
C. Ramires-Neto*, G.A. Monteiro, Y.F.R. Sancler-Silva, R.R.D. Maziero, F.P. Lisboa,C.P. Freitas-Dell’Aqua, M.A. Alvarenga, F.O. PapaDepartment of Animal Reproduction and Veterinary Radiology, School of Veterinary Medicine and Animal Science, UNESP, Botucatu, SP, Brazil
Pasquini et al. (Anim Reprod Sci 107:338-339, 2008)observed that adding sperm motility stimulants improvesmotility of epididymal sperm after freezing and thawing.Morris et al. (Theriogenology 58:643-646, 2002) found animprovement in fertility when cryopreserved epididymalspermwere added to Sperm-TALP prior to freezing. The aimof the present study was to evaluate the effect of differentmotility stimulants on cryopreserved semen from sub-fertile stallions. Six stallions of different breeds with ahistory of low fertility (pregnancy rate < 25%) were used.Semen was collected using an artificial vagina, and semendilution (1:1) was performed with Botu-SemenTM (Botu-pharma, Brazil). After dilution, 20% Botu-SemenTM (groupCG), Sperm-TALP (group GS, Human Tubal Fluid mediumsupplemented with 0.5mg/mL caffeine,100IU/mL penicillinand 100g/mL streptomycin) or Fert-TALP (group GF, HumanTubal Fluid medium supplemented with 0.5mg/mLcaffeine, 0.003 g/mL heparin, 0.0003g/mL penicillamine,0.00011g/mL hypotaurine, 0.00018g/mL epinephrine, 100IU/mL penicillin and 100g/mL streptomycin) was added tothe extended semen. Samples were incubated for 15 min at37�C and then centrifuged at 600xg for 10 min. The su-pernatant was discarded, and the pellet was resuspendedwith Botu-CryoTM at a concentration of 100x106 totalsperm/mL. The semen was packaged in 0.5-mL straws andcryopreserved (sequentially: 5�C/20 min; -120oC/20 min,followed by immersion in liquid nitrogen). The straws werethawed for 20 sec at 46�C. After incubation at 37�C (M1)and after freezing and thawing (M2) the sperm kinetic
* Presenting author
analysis was performed by CASA (HTM-IVOS, IMV, USA). AtM2, plasma membrane integrity (MI, probes propidiumiodide and FITC-PSA), translocation of membrane phos-pholipids (TP, annexin V) and DNA fragmentation assess-ment (DNA, probe Acridine Orange) were determined byflow cytometry (BD LSR Fortessa, BD, Mountain View, USA).All sperm parameters were compared by analysis of vari-ance (ANOVA). The significance level consideredwas 5%. Nodifference was observed (p>0.05) at M1 in total motility(TM%, GC¼24.1�16.1, GS¼26.3�15.8, GF¼29.3�17.8), pro-gressive motility (PM%, GC¼8.5�8.5, GS¼8.3�7.6,GF¼9.5�9.0), progressive angular velocity (VAP, mm/s,GC¼101.3�14.3, GS¼106.6�11.8, GF¼74.8�44.4) andpercentage of rapid sperm (RAP%; GC¼17.5�12.8, GS¼19.6�13.4, GF¼22.1�15.3). At M2 there were no differences(p>0.05) in TM (GC¼16.2�15.0, GS¼19.8�17.4, GF¼16.3�14.7), PM (GC¼7.5�8.5, GS¼10.1�10.5, GF¼7.8�9.7), VAP(GC¼75.8�22.8, GS¼81.0�25.2, GF¼78.8�18.1), RAP(GC¼10.1�11.3, GS¼14.0�14.0, GF¼10.1�11.1), MI (%, GC¼15.1�4.8, GS¼15.2�6.4, GF¼14.5�7.9), TP (%, GC¼6.9�10.7,GS¼4.5�4.5, GF¼3.5�3.2) and DNA (GC¼2.6�0.6, GS¼2.7�0.6, GF¼2.5�0.6) among the groups. In conclusion, therewas no effect of addition of motility stimulators on spermkinetic parameters and flow cytometric parameters forfrozen semen from subfertile stallions.
Acknowledgments
FAPESP.
