Effect of density gradient on sperm parameters of stallion frozen semen
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performed on donkeys. The aim of this study was to determine if such centrifugation of donkey semen after 24 hours of cooled storage improves sperm motility. Semen was collected from four Andalusian donkeys by artificial vagina. Two aliquots of each semen sample were extended with INRA96, cooled slowly to 5 oC and then stored in a styrofoam box. Single layer centrifugation was carried out after 24 hours of cooled storage using the silane-coated silica colloid Androcoll-E Large. Computer assisted sperm analysis (CASA) was performed and sperm motility, total motility and progressive motility (considered on settings when straightened percentage was equal or higher than 75%), were statistically compared between semen aliquots processed or not by colloid centrifugation. Results were expressed as mean standard error. The yield of selected spermatozoa (number of spermatozoa in sperm pellet/ number of spermatozoa in initial load) was 12.94 1.10%. Significant differences (P < 0.05) were found between colloid-selected and unselected sperm samples for progressive sperm motility (52.6 7.1% vs 30.2 5.8%). However, no significant differences were found for total sperm motility (77.7 6.4% vs 64.6 8.1%). These results suggest an additional option for improving sperm motility in donkey semen. However, these are preliminary results and further studies should be performed increasing the number of animals and the semen parameters assessed. In conclusion, centrifugation through a single layer of colloid after 24 h of cooled storage increased progressive sperm motility in a limited number of donkey semen samples. Reference [1] Morrell al. Prolongation of stallion sperm survival by centrifugation through coated silica colloids: a preliminary study. Animal Repro- duction 2008;5:121-6. Effect of density gradient on sperm parameters of stallion frozen semen P.M. Papa, R.R.D. Maziero, F.P. Hartwig, F.P. Lisboa, J.A. Delláqua, Jr., M.A. Alvarenga, P.N. Guasti, and F.O. Papa Department of Animal Reproduction and Veterinary Radiology, School of Veterinary Medicine and Animal Science, UNESP, Botucatu, SP 18610-970, Brazil The objective of this study was to evaluate the influence of density gradient centrifugation EquipureÔ on sperm parameters of stallion frozen semen. For this experiment, two ejaculates from nine stallions were divided into two groups: Control and Treatment. In the Control group, the sperm concentration was 1x109 then the samples were diluted with a skim milk-based extender (Botu-SêmenÔ) (v:v), centrifuged at 600xg for 10 min and resuspended in Botu-CrioÔ freezing extender at concentration of 200x106spermatozoa/mL and the recovered rate was 90% of total sperm. In Treatment group, the sperm concentra- tion was 1x109 and then samples were diluted with Botu- SêmenÔ and centrifuged at 600xg for 10 minutes, the supernatant was removed and the pellet was resuspended in 5mL of Botu-SêmenÔ. After this procedure, 5mL of density gradient was placed in a 15mL tube. Then, the tube was slightly inclined and 5mL of sperm previously resus- pended with Botu-SêmenÔ was slowly added. The samples were centrifuged at 400xg for 20 minutes and the filtered pellet was recovered with a 1000mL pipette. The recovered rate was 41% of viable sperm. The samples were then added to Botu-CrioÔ and 0.5 mL straws were filled with 100x106 sperm, and then submitted to freezing process. The straws were thawed at 46 o C for 20 seconds and semen samples were evaluated before freezing and after thawing by CASA for total motility (TM), progressive motility (PM), rapid sperm (RAP) and plasma membrane integrity (PMI) by epifluorescent microscopy. All parameters were analyzed by ANOVA, followed by Tukey's test to identify the signifi- cant differences (P < 0.05). Both pre-freeze and post-thaw, a superiority was observed in Treatment group for parameters of TM, PM and RAP when compared to the Control group. No differences in plasma membrane integ- rity were observed between the groups (P < 0.05). Acknowledgments FAPESP for financial support and Botupharma for the donation of Botu-SêmenÔ and Botu-CrioÔ extender. Raw semen concentration directly influences CASA velocity pathways S. Parrilla-Hernandez 1, 2 , S. Deleuze 1, 2 , F. Van Den Berghe 1 , and J. Ponthier 1, 2 1 Equine Clinic, Veterinary Medicine Faculty, University of Liège, B-4000, Liège, Belgium, 2 linalux-Mls, Centre Européen du Cheval, 18 rue des Champs Elysées, B-5590, Ciney, Belgium Physiologically, raw semen characteristics are influenced by stallion and semen conditions. The aim of this study was to investigate how computer-assisted semen analysis (CASA) results are influenced by raw semen characteristics. One hundred semen collections (10 collections per stallion, 3 Arabian and 7 sport horses between 3 and 23 years old) were analysed. Number of mounts before ejaculation and semen volume were recorded. Spermatozoa concentration was determined using a NucleoCounter SP100 (Allerod). Motility pathways from semen re-diluted to 20x10 6 spz/ml Table 1 Mean values of sperm parameters of total motility (TM), progressive motility (PM), percentage of rapid sperm (RAP) and plasma membrane integrity (PMI) in pre-freeze and post-thaw semen samples for Control and Treatment groups Pre-freeze Post-thaw Control Treatment Control Treatment TM (%) 75 b 86 a 54 B 66 A PM (%) 30 b 45 a 20 B 34 A RAP (%) 65 b 79 a 34 B 45 A PMI (%) 59 b 68 a 40 A 47 A The findings in the present study show that the centrifugation of semen with the density gradient improves the freezability of equine sperm. This gradient may be an important tool to improve sperm parameters and possibly the fertility of “bad freezer” stallions. a,b Different letters in a row indicate differences (P < 0.05). A,B Different letters in a row indicate differences (P < 0.05). 6 th ISSR Abstracts / Journal of Equine Veterinary Science 32 (2012) 475-518 505
Transcript of Effect of density gradient on sperm parameters of stallion frozen semen
Table 1Mean values of sperm parameters of total motility (TM), progressivemotility (PM), percentage of rapid sperm (RAP) and plasma membraneintegrity (PMI) in pre-freeze and post-thaw semen samples for Controland Treatment groups
Pre-freeze Post-thaw
Control Treatment Control Treatment
TM (%) 75b 86a 54B 66A
PM (%) 30b 45a 20B 34A
RAP (%) 65b 79a 34B 45A
6th ISSR Abstracts / Journal of Equine Veterinary Science 32 (2012) 475-518 505
performed on donkeys. The aim of this study was todetermine if such centrifugation of donkey semen after 24hours of cooled storage improves sperm motility. Semenwas collected from four Andalusian donkeys by artificialvagina. Two aliquots of each semen sample were extendedwith INRA96, cooled slowly to 5 oC and then stored ina styrofoam box. Single layer centrifugationwas carried outafter 24 hours of cooled storage using the silane-coatedsilica colloid Androcoll-E Large. Computer assisted spermanalysis (CASA) was performed and sperm motility, totalmotility and progressive motility (considered on settingswhen straightened percentage was equal or higher than75%), were statistically compared between semen aliquotsprocessed or not by colloid centrifugation. Results wereexpressed as mean � standard error. The yield of selectedspermatozoa (number of spermatozoa in sperm pellet/number of spermatozoa in initial load) was 12.94 � 1.10%.Significant differences (P < 0.05) were found betweencolloid-selected and unselected sperm samples forprogressive sperm motility (52.6 � 7.1% vs 30.2 � 5.8%).However, no significant differences were found for totalsperm motility (77.7 � 6.4% vs 64.6 � 8.1%). These resultssuggest an additional option for improving sperm motilityin donkey semen. However, these are preliminary resultsand further studies should be performed increasing thenumber of animals and the semen parameters assessed. Inconclusion, centrifugation through a single layer of colloidafter 24 h of cooled storage increased progressive spermmotility in a limited number of donkey semen samples.
PMI (%) 59b 68a 40A 47A
The findings in the present study show that the centrifugation of semenwith the density gradient improves the freezability of equine sperm. Thisgradient may be an important tool to improve sperm parameters andpossibly the fertility of “bad freezer” stallions.a,bDifferent letters in a row indicate differences (P < 0.05).A,B
Reference
[1] Morrell al. Prolongation of stallion sperm survival by centrifugationthrough coated silica colloids: a preliminary study. Animal Repro-duction 2008;5:121-6.
Different letters in a row indicate differences (P < 0.05).
Effect of density gradient on sperm parameters ofstallion frozen semen
P.M. Papa, R.R.D. Maziero, F.P. Hartwig, F.P. Lisboa, J.A.Delláqua, Jr., M.A. Alvarenga, P.N. Guasti, and F.O. PapaDepartment of Animal Reproduction and VeterinaryRadiology, School of Veterinary Medicine and AnimalScience, UNESP, Botucatu, SP 18610-970, Brazil
The objective of this study was to evaluate the influence ofdensity gradient centrifugation Equipure� on spermparameters of stallion frozen semen. For this experiment,two ejaculates from nine stallions were divided into twogroups: Control and Treatment. In the Control group, thesperm concentration was 1x109 then the samples werediluted with a skim milk-based extender (Botu-Sêmen�)(v:v), centrifuged at 600xg for 10 min and resuspended inBotu-Crio� freezing extender at concentration of200x106spermatozoa/mL and the recovered rate was 90%of total sperm. In Treatment group, the sperm concentra-tion was 1x109 and then samples were diluted with Botu-Sêmen� and centrifuged at 600xg for 10 minutes, thesupernatant was removed and the pellet was resuspendedin 5mL of Botu-Sêmen�. After this procedure, 5mL ofdensity gradient was placed in a 15mL tube. Then, the tube
was slightly inclined and 5mL of sperm previously resus-pended with Botu-Sêmen�was slowly added. The sampleswere centrifuged at 400xg for 20 minutes and the filteredpellet was recovered with a 1000mL pipette. The recoveredrate was 41% of viable sperm. The samples were then addedto Botu-Crio� and 0.5 mL straws were filled with 100x106sperm, and then submitted to freezing process. The strawswere thawed at 46oC for 20 seconds and semen sampleswere evaluated before freezing and after thawing by CASAfor total motility (TM), progressive motility (PM), rapidsperm (RAP) and plasma membrane integrity (PMI) byepifluorescent microscopy. All parameters were analyzedby ANOVA, followed by Tukey's test to identify the signifi-cant differences (P < 0.05). Both pre-freeze and post-thaw,a superiority was observed in Treatment group forparameters of TM, PM and RAP when compared to theControl group. No differences in plasma membrane integ-rity were observed between the groups (P < 0.05).
Acknowledgments
FAPESP for financial support and Botupharma for thedonation of Botu-Sêmen� and Botu-Crio� extender.
Raw semen concentration directly influences CASAvelocity pathways
S. Parrilla-Hernandez 1,2, S. Deleuze 1,2,F. Van Den Berghe 1, and J. Ponthier 1,21 Equine Clinic, Veterinary Medicine Faculty, University ofLiège, B-4000, Liège, Belgium, 2 linalux-Mls, CentreEuropéen du Cheval, 18 rue des Champs Elysées, B-5590,Ciney, Belgium
Physiologically, raw semen characteristics are influencedby stallion and semen conditions. The aim of this study wasto investigate how computer-assisted semen analysis(CASA) results are influenced by raw semen characteristics.One hundred semen collections (10 collections per stallion,3 Arabian and 7 sport horses between 3 and 23 years old)were analysed. Number of mounts before ejaculation andsemen volume were recorded. Spermatozoa concentrationwas determined using a NucleoCounter SP100 (Allerod).Motility pathways from semen re-diluted to 20x106 spz/ml
