title: Detrimental effects of cryopreservation of loach (Misgurnus fossilis) sperm on subsequent embryo development are reversed by incubating fertilised eggs in caffeine.
abstract: Cryopreservation can cause changes to the genetic material of cells, but the mechanism and significance of these changes are still unknown. It has been suggested that some damage to the sperm genome could be repaired by the DNA repair system of the oocyte after fertilisation. Caffeine has been reported to be an inhibitor of such repair processes. In this study the effect of caffeine on the repair system of Loach (Misgurnus fossilis) oocytes was investigated. Loach eggs were fertilised using cryopreserved sperm. Embryos derived from cryopreserved sperm were exposed to 2.6mM caffeine for 1h after fertilisation. The experiments were carried out using 32313 embryos from four females and eight males. Embryo survival was evaluated for 46 h until the hatching stage. Reduction in embryo survival after 20th stage is generally believed to result from the failure in the genome function of embryos. Cryopreservation of sperm significantly decreased embryo survival (53.4+/-2.8% compared to 68.4+/-2.8% of control) after the 20th stage. However, the addition of caffeine to the embryos derived from cryopreserved sperm, in contrast to our expectation, significantly increased survival of loach embryos (70.9+/-2.8% compared to 53.4+/-2.8% of embryos derived from cryopreserved sperm in the absence of caffeine). The effect of individual donors of sperm and eggs on overall embryo survival was also studied. Whilst no significant differences were observed between males, the effect of individual females on embryo survival was significant. The analysis of embryo survival at different developmental stages showed that embryo survival both before and after 20th stage decreased with embryo development. When fresh sperm were used the decline of embryo survival with development was more pronounced compared with those embryos derived from cryopreserved sperm. Possible explanations of these effects are presented.
authors.affiliation: Luton Institute of Research in the Applied Natural Sciences, University of Luton, The Spires, 2 Adelaide Street, Luton, LU1 5DU, UK.
authors.forename: J
authors.initials: J
authors.lastname: Kopeika
authors.forename: E
authors.initials: E
authors.lastname: Kopeika
authors.forename: T
authors.initials: T
authors.lastname: Zhang
authors.forename: D M
authors.initials: DM
authors.lastname: Rawson
authors.forename: W V
authors.initials: WV
authors.lastname: Holt
mesh_terms: D000818:Animals; D002110:Caffeine / Q000494:pharmacology*; D015925:Cryopreservation; D003451:Cryoprotective Agents / Q000633:toxicity; D003470:Culture Media / Q000494:pharmacology*; D003531:Cypriniformes / Q000196:embryology*; D004249:DNA Damage; D004260:DNA Repair / Q000187:drug effects*; D004305:Dose-Response Relationship, Drug; D004625:Embryo, Nonmammalian / Q000187:drug effects / Q000502:physiology; D005260:Female; D005307:Fertilization in Vitro; D008297:Male; D009865:Oocytes / Q000187:drug effects* / Q000502:physiology; D012662:Semen Preservation / Q000009:adverse effects*; D013094:Spermatozoa / Q000187:drug effects*; D015053:Zygote / Q000187:drug effects* / Q000502:physiology
chemical_list: D003451:Cryoprotective Agents; D003470:Culture Media; D002110:Caffeine