Comparison of sperm parameters and fertility between sperm cryopreserved from ejaculated semen and...
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Comparison of sperm parameters and fertility between sperm cryopreserved from ejaculated semen and from the epididymis of subfertile stallions G.A. Monteiro * , Y.F.R. Sancler-Silva, C. Ramires-Neto, C.P. Freitas-Dell’Aqua, F.P. Hartwig, I. Martin, A.M. Crespilho, M.A. Alvarenga, F.O. Papa Department of Animal Reproduction and Veterinary Radiology, School of Veterinary Medicine and Animal Science, UNESP, Botucatu, Brazil Despite the benefits provided by seminal plasma during natural cover, its function is questionable when a biotech- nology such artificial insemination is used. Studies employing spermatozoa from the cauda epididymis with or without the addition of seminal plasma showed no differ- ence in conception rates. Furthermore, studies have re- ported deleterious effects of secretions from accessory glands on sperm viability and conception rate (Aurich et al., Theriogenology, 1996; 46, 791-797), especially in stallions with poor semen quality. Thus the aims of this study were: 1) compare the sperm kinetic parameters, viability and fertility of sperm recovered from the ejaculate (G1) and from the cauda epididymis (G2) of subfertile stallions (pregnancy rate<35%). For G1, one ejaculate from each of eight subfertile stallions was collected and then subjected to cryopreservation using the extender BotuCrio TM . One week after the last semen collection, the stallions under- went bilateral orchiectomy. Sperm from the cauda epidid- ymis were harvested immediately after castration by retrograde flushing the caudal portion (G2) of the epidid- ymis using a skim milk-based extender (BotuSemen TM ). The recovered sperm were then cryopreserved using BotuCrio TM extender. The sperm samples were analyzed for kinetic parameters by computer-assisted analyses (CASA- HTM-IVOS) and plasma membrane integrity (Propidium iodide, Sigma; Hoechst 33345, Sigma), acrosome integrity (FITC-PSA, Sigma; Hoechst 33345, Sigma) and degree of translocation of phosphatidylserine (Annexin-V–APC, Pharmingen; Hoechst 33345, Sigma) by flow cytometry (BD-LSRFortessaÔ). For analysis, straws were thawed in a 46 C water bath for 20 sec. Artificial insemination was performed in the tip of the uterine horn ipsilateral to ovulation using a flexible pipette containing 800 x 10 6 motile frozen-thawed sperm, corresponding to a pool of eight straws from different stallions. The statistical analysis was performed using ANOVA using Proc GLM 9.2 (SAS Institute, USA). The general linear model analysis of vari- ance was used to identify differences in fertility rates ac- cording to the treatment, and they were separated using Student-Newman-Kuels. The level of statistical significance was defined as p <0.05. The sperm kinetic parameters of the G1 vs G2 samples were 13.1 10.2 a vs 32.513.0 b , 4.43.6 a vs 12.28.9 b and 7.25.6 a vs 19.411.6 b for total motility (TM), progressive motility (PM) and percentage of rapid cells (RAP), respectively. The sperm viability param- eters of the G1 vs G2 samples were 18.57.6 a vs 38.99.6 b , 52.411.2 a vs 73.47.6 b and 2.52.6 a vs 1.92.1 b for plasma membrane integrity (PMI), acrosome integrity (ACI) and degree of translocation of phosphatidylserine (ANEX), respectively. After artificial insemination, fertility for G2 (48.4%, 15/31) was higher than G1 (22.6%, 7/31). These re- sults allow us to conclude that sperm recovered from the cauda epididymis of subfertile stallions is more resistant to freezing when compared to the ejaculated sperm. The epididymal sperm after thawing also showed improved sperm kinetic parameters (TM, PM, RAP), viability (PMI, ACI and ANEX) and fertility when compared to ejaculated sperm. The lack of exposure of sperm recovered from the cauda epididymis to seminal plasma may be a possible cause of the improved viability found in this study. 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) 71
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Journal of Equine Veterinary Science 34 (2014) 71
Contents lists ava
Journal of Equine Veterinary Science
journal homepage: www.j -evs.com
Comparison of sperm parameters and fertility betweensperm cryopreserved from ejaculated semen and from theepididymis of subfertile stallions
G.A. Monteiro*, Y.F.R. Sancler-Silva, C. Ramires-Neto, C.P. Freitas-Dell’Aqua, F.P. Hartwig,I. Martin, A.M. Crespilho, M.A. Alvarenga, F.O. PapaDepartment of Animal Reproduction and Veterinary Radiology, School of Veterinary Medicine and Animal Science, UNESP, Botucatu, Brazil
Despite the benefits provided by seminal plasma duringnatural cover, its function is questionable when a biotech-nology such artificial insemination is used. Studiesemploying spermatozoa from the cauda epididymis with orwithout the addition of seminal plasma showed no differ-ence in conception rates. Furthermore, studies have re-ported deleterious effects of secretions from accessoryglands on sperm viability and conception rate (Aurich et al.,Theriogenology, 1996; 46, 791-797), especially in stallionswith poor semen quality. Thus the aims of this study were:1) compare the sperm kinetic parameters, viability andfertility of sperm recovered from the ejaculate (G1) andfrom the cauda epididymis (G2) of subfertile stallions(pregnancy rate<35%). For G1, one ejaculate from each ofeight subfertile stallions was collected and then subjectedto cryopreservation using the extender BotuCrioTM. Oneweek after the last semen collection, the stallions under-went bilateral orchiectomy. Sperm from the cauda epidid-ymis were harvested immediately after castration byretrograde flushing the caudal portion (G2) of the epidid-ymis using a skim milk-based extender (BotuSemenTM).The recovered sperm were then cryopreserved usingBotuCrioTM extender. The sperm samples were analyzed forkinetic parameters by computer-assisted analyses (CASA-HTM-IVOS) and plasma membrane integrity (Propidiumiodide, Sigma; Hoechst 33345, Sigma), acrosome integrity(FITC-PSA, Sigma; Hoechst 33345, Sigma) and degree oftranslocation of phosphatidylserine (Annexin-V–APC,Pharmingen; Hoechst 33345, Sigma) by flow cytometry(BD-LSRFortessa�). For analysis, straws were thawed in a46�C water bath for 20 sec. Artificial insemination wasperformed in the tip of the uterine horn ipsilateral to
* Presenting author
ovulation using a flexible pipette containing 800 x 106
motile frozen-thawed sperm, corresponding to a pool ofeight straws from different stallions. The statistical analysiswas performed using ANOVA using Proc GLM 9.2 (SASInstitute, USA). The general linear model analysis of vari-ance was used to identify differences in fertility rates ac-cording to the treatment, and they were separated usingStudent-Newman-Kuels. The level of statistical significancewas defined as p <0.05. The sperm kinetic parameters ofthe G1 vs G2 samples were 13.1�10.2a vs 32.5�13.0b,4.4�3.6a vs 12.2�8.9b and 7.2�5.6a vs 19.4�11.6b for totalmotility (TM), progressive motility (PM) and percentage ofrapid cells (RAP), respectively. The sperm viability param-eters of the G1 vs G2 samples were 18.5�7.6a vs 38.9�9.6b,52.4�11.2a vs 73.4�7.6b and 2.5�2.6a vs 1.9�2.1b for plasmamembrane integrity (PMI), acrosome integrity (ACI) anddegree of translocation of phosphatidylserine (ANEX),respectively. After artificial insemination, fertility for G2(48.4%, 15/31) was higher than G1 (22.6%, 7/31). These re-sults allow us to conclude that sperm recovered from thecauda epididymis of subfertile stallions is more resistant tofreezing when compared to the ejaculated sperm. Theepididymal sperm after thawing also showed improvedsperm kinetic parameters (TM, PM, RAP), viability (PMI, ACIand ANEX) and fertility when compared to ejaculatedsperm. The lack of exposure of sperm recovered from thecauda epididymis to seminal plasma may be a possiblecause of the improved viability found in this study.
Acknowledgments
FAPESP
