DNA integrity of stallion spermatozoa from different segments of the epididymis
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DNA integrity of stallion spermatozoa from different segments of the epididymis C.M. Melo-Oña * , L.C.O. Magalhães, C.P. Freitas-Dell’Aqua, F.O. Papa Department of Animal Science, UFG, Goiânia Brazil, Department of Animal Reproduction, Unesp, Botucatu, Brazil The cryopreservation of sperm from different segments of the epididymis is the ultimate opportunity to preserve the gene pool of animals of great breeding value. Despite the fact that samples from the caput epididymis are immotile, their DNA integrity could justify their use in ICSI procedures. Thus, the objective of the present study was to evaluate the DNA integrity and sperm motility of stallion sperm collected from different regions of the epididymis. Equine epididymides were obtained from 10 stallions using retrograde flush with 40mL of Botu-Semen per caudae epididymis. Sperm from the caudae epididymis were obtained with a slicing technique in association with a float-up method. The samples were divided into 3 groups: Botu-SemenÒ (control), Fert-Talp and Sperm-Talp, and diluted 1:1 with these media. The samples were centrifuged (1000 x g/10 min) to concentrate the sperm, the supernatant was removed, and the pellets were resuspended in Botu-CrioÒ. Samples were loaded into 0.5- mL straws and cooled at 5 C for 20 min. They were then frozen in nitrogen vapor (6 cm above the level of liquid nitrogen) for an additional 20 min, plunged into liquid nitrogen and stored. Sperm chromatin structure assay was performed using a semen aliquot diluted in 200 mL of buffer solution (0.186g disodium EDTA, 0.790 g Tris-HC1, 4.380 g NaC1 in 500 mL deionized water, pH 7.4). This was added with 400 mL of detergent/acid solution. Thirty sec- onds later, 1.2 mL acridine orange solution was added (Evenson, In: Sorsa M, Norppa H (ed), Monitoring of Occupational Genotoxicants. Alan R Liss, p.121-132, 1986). Flow cytometry was performed within 5 min of acridine orange addition, and data were analyzed using the WinList 6.0 software according to Flaherty et al. (Hum. Reprod. (2008) 23: 1044-1052). An analysis of variance and Tukey’s test with a P < 0.05 significance level were used to compare the mean values. There was no difference among media within the epidididymal segments for all parame- ters. Spermatozoa from the caput epididymis had no motility. Sperm motility for the corpus epididymis was: (total motility: 18.213.24; 34.016.15 and 38.324.88) and progressive motility: 5.95.16; 14.98.85 and 15.312.03) for BS, Fert-talp and Sperm-Talp, respectively. Sperm motility for caudae epididymis was: (total motility: 36.218.18; 52.318.40 and 51.422.22) and progressive motility (13.89.27; 27.713.48 and 28.1 15.29) were assessed for BS, Fert-talp and Sperm-Talp samples, respectively. Mean DNA fragmentation indices (SD) for Botu-Semen, sperm-TALP and fert-TALP in sperm from the caput epididymis (7.94.7; 7.04.3 and 9.84.4), corpus epididymis (4.06.1; 1.91.6 and 2.32.5) and cauda epididymis (1.81.7; 1.42.0 and 1.32.5), respectively. The present study did not find any sample above 10% DNA fragmentation in any of the epididymal segments. Like- wise, the three media were efficient in maintaining sperm DNA integrity. Thus, based on the results of the present study, we can conclude that all three media can be used to incubate epidydimal spermatozoa before freezing. * 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) 95
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Journal of Equine Veterinary Science 34 (2014) 95
Contents lists ava
Journal of Equine Veterinary Science
journal homepage: www.j -evs.com
DNA integrity of stallion spermatozoa from differentsegments of the epididymis
C.M. Melo-Oña*, L.C.O. Magalhães, C.P. Freitas-Dell’Aqua, F.O. PapaDepartment of Animal Science, UFG, Goiânia Brazil, Department of Animal Reproduction, Unesp, Botucatu, Brazil
The cryopreservation of sperm from different segmentsof the epididymis is the ultimate opportunity to preservethe gene pool of animals of great breeding value. Despitethe fact that samples from the caput epididymis areimmotile, their DNA integrity could justify their use in ICSIprocedures. Thus, the objective of the present study was toevaluate the DNA integrity and sperm motility of stallionsperm collected from different regions of the epididymis.Equine epididymides were obtained from 10 stallionsusing retrograde flush with 40mL of Botu-Semen percaudae epididymis. Sperm from the caudae epididymiswere obtained with a slicing technique in association witha float-up method. The samples were divided into 3groups: Botu-Semen� (control), Fert-Talp and Sperm-Talp,and diluted 1:1 with these media. The samples werecentrifuged (1000 x g/10 min) to concentrate the sperm,the supernatant was removed, and the pellets wereresuspended in Botu-Crio�. Samples were loaded into 0.5-mL straws and cooled at 5�C for 20 min. They were thenfrozen in nitrogen vapor (6 cm above the level of liquidnitrogen) for an additional 20 min, plunged into liquidnitrogen and stored. Sperm chromatin structure assay wasperformed using a semen aliquot diluted in 200 mL ofbuffer solution (0.186g disodium EDTA, 0.790 g Tris-HC1,4.380 g NaC1 in 500 mL deionized water, pH 7.4). This wasadded with 400 mL of detergent/acid solution. Thirty sec-onds later, 1.2 mL acridine orange solution was added(Evenson, In: Sorsa M, Norppa H (ed), Monitoring of
* Presenting author
Occupational Genotoxicants. Alan R Liss, p.121-132, 1986).Flow cytometry was performed within 5 min of acridineorange addition, and data were analyzed using the WinList6.0 software according to Flaherty et al. (Hum. Reprod.(2008) 23: 1044-1052). An analysis of variance andTukey’s test with a P < 0.05 significance level were used tocompare the mean values. There was no difference amongmedia within the epidididymal segments for all parame-ters. Spermatozoa from the caput epididymis had nomotility. Sperm motility for the corpus epididymis was:(total motility: 18.2�13.24; 34.0�16.15 and 38.3�24.88)and progressive motility: 5.9�5.16; 14.9�8.85 and15.3�12.03) for BS, Fert-talp and Sperm-Talp, respectively.Sperm motility for caudae epididymis was: (total motility:36.2�18.18; 52.3�18.40 and 51.4�22.22) and progressivemotility (13.8�9.27; 27.7�13.48 and 28.1�15.29) wereassessed for BS, Fert-talp and Sperm-Talp samples,respectively. Mean DNA fragmentation indices (�SD) forBotu-Semen, sperm-TALP and fert-TALP in sperm from thecaput epididymis (7.9�4.7; 7.0�4.3 and 9.8�4.4), corpusepididymis (4.0�6.1; 1.9�1.6 and 2.3�2.5) and caudaepididymis (1.8�1.7; 1.4�2.0 and 1.3�2.5), respectively.The present study did not find any sample above 10% DNAfragmentation in any of the epididymal segments. Like-wise, the three media were efficient in maintaining spermDNA integrity. Thus, based on the results of the presentstudy, we can conclude that all three media can be used toincubate epidydimal spermatozoa before freezing.
