title: Alterations of sperm DNA integrity during cryopreservation procedure and in vitro incubation of bull semen.
abstract: An association between sperm DNA integrity and fertility was recently shown for frozen-thawed Norwegian Red (NRF) bull semen diluted in skimmed milk egg yolk (SMEY). In general the fertility of NRF cattle is high, however, in comparison with NRF semen in SMEY, NRF semen diluted in Tris EY based extenders has shown reduced fertility. The aim of the present study was to do a split-sample comparison of sperm DNA integrity of NRF bull semen (n=20) in SMEY and Triladyl (Tris EY based) during routine cryopreservation procedure and during in vitro incubation of frozen-thawed semen in modified synthetic oviduct fluid (mSOF). In contrast to the high fertility of NRF cattle, Holstein cattle are experiencing a marked decline in fertility. Therefore, the present study also aimed to compare sperm DNA integrity of NRF (n=20) and Holstein (n=20) semen diluted in Triladyl during in vitro incubation. The sperm DNA integrity was measured by susceptibility to in situ acid induced denaturation by the Sperm chromatin structure assay (SCSA). Compared to initial values of frozen neat semen, an increase in DNA damage was observed after dilution and cooling (5 degrees C) and after freezing-thawing of NRF semen in SMEY, but only after freezing-thawing for NRF semen diluted in Triladyl. Sperm DNA damage of NRF semen increased during in vitro incubation in mSOF; the increase in percentage of spermatozoa with DNA damage was more prominent in SMEY than in Triladyl, while the degree of damage was higher in Triladyl, throughout the incubation period. However, while the correlation between DNA damage and sperm survival was negative in SMEY throughout the incubation period, a positive correlation was observed in Triladyl after 9h of incubation, indicating a higher presence of DNA damage in the live sperm population. In comparison with Holstein spermatozoa, the sperm DNA integrity of NRF semen reflected a better ability to withstand alterations induced during in vitro incubation in mSOF. In conclusion, sperm DNA integrity of NRF bull semen was altered during the cryopreservation procedure and in vitro incubation in mSOF. Dilution in Triladyl maintained bull sperm DNA integrity better than dilution in SMEY. Furthermore, alterations in Holstein sperm DNA integrity was more pronounced during in vitro incubation in mSOF compared to NRF bull spermatozoa.
authors.affiliation: Team Semin, P.O. Box 8146 Dep., N-0033 Oslo, Norway. karin.waterhouse@veths.no
authors.forename: K E
authors.initials: KE
authors.lastname: Waterhouse
authors.forename: A
authors.initials: A
authors.lastname: Gjeldnes
authors.forename: A
authors.initials: A
authors.lastname: Tverdal
authors.forename: P M
authors.initials: PM
authors.lastname: De Angelis
authors.forename: W
authors.initials: W
authors.lastname: Farstad
authors.forename: M
authors.initials: M
authors.lastname: Håård
authors.forename: E
authors.initials: E
authors.lastname: Kommisrud
mesh_terms: D000818:Animals; D001826:Body Fluids; D002417:Cattle; D002462:Cell Membrane / Q000648:ultrastructure; D015925:Cryopreservation / Q000379:methods / Q000662:veterinary*; D004247:DNA / Q000737:chemistry*; D004249:DNA Damage; D005187:Fallopian Tubes; D005260:Female; D006358:Hot Temperature; D008297:Male; D012661:Semen / Q000166:cytology / Q000502:physiology*; D012662:Semen Preservation / Q000379:methods / Q000662:veterinary*; D013094:Spermatozoa / Q000737:chemistry* / Q000648:ultrastructure
chemical_list: D004247:DNA