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 different historiy 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.

DNA integrity of fresh and frozen canine epididymal spermatozoa

“…The SCD test was reported to be simple, highly reproducible and inexpensive technique, and the SCD results correlated with the SCSA results [28]. In the current paper, the DNA fragmentation indices were similar in fresh and frozen canine epididymal spermatozoa, which confirms the resilience of canine ejaculated sperm DNA to cold stress [12,15,16]. A progressive deterioration of DNA has been observed after exposure of fresh or thawed spermatozoa to different stressors (e.g., incubation time and temperature, toxicants, ROS) [21,29], indicating that thefreezing/thawing procedure facilitates the destabilization of the chromatin structure of spermatozoa [21].…”

“…The impact of cryopreservation on sperm DNA integrity is still a controversial matter in mammals including dogs. Some authors showed that the freezing/thawing procedure did not produce significant adverse effects on chromatin status in canine ejaculated spermatozoa [12,15,16]. In contrast, Kim et al [14] found a higher DNA fragmentation in thawed compared to fresh spermatozoa.…”

“…Sperm DNA integrity has been evaluated in fresh ejaculated [7][8][9][10] and epididymal canine semen [11]. Few reports have compared fresh and post-thaw chromatin integrity of canine ejaculated spermatozoa, obtaining variable results [12][13][14][15][16], but the post-thaw DNA stability of canine epididymal spermatozoa has not been investigated. The integrity of the paternal DNA is of crucial importance for embryo development [17], and a relationship between DNA damage and infertility has been demonstrated in humans.…”

Amphibian sperm DNA fragmentation

“…Not surprisingly, there was little difference between values of SDF obtained at the onset of incubation for fresh and frozen-thawed samples (either DMSO or SAR). Similar findings have been reported in stallion , ram , dog (Prinosilova et al 2012), koala (Johnston et al 2012b) and rainbow trout (Labbe et al 2001). Although the chromatin structure may appear unaltered by the cryopreservation process immediately upon thawing, the varying resistance of sperm DNA to stressors associated with post-thaw incubation ex vivo, particularly influences the longevity of sperm DNA quality (Gosálvez et al 2011a).…”

“…Sperm cryopreservation protocols that involve penetrating cryoprotectants developed for other amphibian species (Beesley et al 1998;Browne et al 1998;Costanzo et al 1998) samples showed a significant increase in SDF after 60 minutes of incubation, the proportion of sperm with SDF was still low, being less than 10%. Similarly, spermatozoa of numerous mammals including dog (Prinosilova et al 2012) and stallion ) retain a normal chromatin structure immediately after thawing (Gosálvez et al 2011b). Although Morrow et al…”