DNA integrity in fresh, chilled and frozen-thawed canine spermatozoa

Abstract

Sperm chromatin status in fresh dog semen and the effect of long-term storage of chilled and frozen dog semen on sperm chromatin integrity was assessed by the sperm chromatin structure assay (SCSA). In the first experiment, the chromatin integrity of fresh semen from 60 dogs with differing histories of fertility was compared with other sperm parameters (total sperm count, sperm motility, viability, acrosomal integrity and sperm morphology). Except for 15 dogs that had never mated before, all were used in breeding as semen donors. Thirty-three of them were successful breeding males while in 12 repeated fertility problems were noted. Ejaculates were assigned to groups with good and poor quality, based on determined sperm motility and percentage of morphologically normal sperm. In the second experiment, chromatin status was measured in fresh and chilled spermatozoa (on day 0, 2, 4, 6, 8 and 10 of storage). Finally, in the third experiment, the chromatin status was measured in fresh and cryopreserved spermatozoa. Evaluating fresh dog semen, we observed that DNA fragmentation index (DFI) and percentage of cells with high DNA stainability (HDS) negatively correlated with total sperm count, percentage of total and progressively motile spermatozoa, sperm viability and percentage of morphologically normal spermatozoa, even with rather low correlation indices. Lower chromatin integrity was found in the group of dog ejaculates showing poor quality in comparison with the group of good quality ejaculates. All dogs with repeated fertility problems were classed in the group showing poor quality, and even though their DFI was significantly higher than the DFI of successful breeding males, the highest DFI we obtained was only just below 9%. We can assume that the chromatin damage level in any of the evaluated dogs was not high enough to have a significant effect on their fertility. Concerning the potential cause of reduced male fertility, the assessment of chromatin integrity in fresh dog ejaculates failed to add any additional information to the results obtained by other techniques of semen analysis. Thus, the current study indicates that neither 10-day preservation of canine sperm chilled in commercial extenders nor long-term cryopreservation in extenders recommended for canine sperm preservation produce adverse effects on sperm chromatin integrity.