82 DNA FRAGMENTATION DYNAMICS OF KOALA SPERMATOZOA

Abstract

Koala sperm chromatin has a tendency to relax following incubation and thawing but the background incidence and dynamics of DNA fragmentation during semen processing at 35°C and following chilled and frozen preservation has not been investigated. This study (n = 10) was designed to establish the fragmentation dynamics of koala sperm DNA at body temperature (35°C), after chilling (4°C) for upward of 16 days, and following a standard freeze–thaw protocol (Johnston SD et al. 2006 Cryobiology 53, 218–228). Sperm DNA fragmentation index (sDFI) was determined using a Halomax kit (ChromaCell SL, Madrid, Spain), which had been customized and validated for koalas (Johnston SD et al. 2007 J. Androl. 28, 891–899). All semen was assessed for sDFI over a 48-h incubation period (T0, T2, T6, T24, and T48) at 35°C. After incubation at 35°C for 48 h, the sDFI and rate of DNA degradation of freshly diluted spermatozoa were highly variable between individuals; the sDFI for 2 koalas remained consistently low (≤2%) whereas the other 8 had sDFI of 8 to 12% after incubation. Chilled storage increased sDFI in all animals, but the rate of increase and the time at which the DNA started to fragment also varied between koalas; sDFI for 1 koala increased immediately upon rewarming after being chilled for 4 h, whereas that of another koala did not increase until after 8 days of chilling and 24 h of incubation at 35°C. Animals also responded to cryopreservation differently; sDFI increased after thawing for 2 of the koalas but did not increase in the others. Subsequent evaluation of frozen–thawed spermatozoa from a greater number of captive koalas (n = 22), and under extended conditions of post-thaw incubation (up to 17 days at 35°C) permitted categorization of the koalas into 3 distinctive groups based on their DNA fragmentation dynamics and rate of DNA degradation. For 7 of the animals, sDFI remained close to the basal level when incubated at 35°C over 7 days, whereas 2 of the koalas had sDFI ranging from 40 to 70% after 24 h of incubation. This study confirmed the occurrence of inter-animal variability in the dynamics of DNA fragmentation, a finding that was apparent whether or not the spermatozoa had been subjected to chilling or cryopreservation.