DNA integrity of canine spermatozoa during chill storage assessed by the sperm chromatin dispersion test using bright-field or fluorescence microscopy.

The objective of this study was to evaluate the effect of chill storage on canine sperm DNA fragmentation assessed by the sperm chromatin dispersion test using bright-field microscopy with Wright solution (sDF-B) or fluorescence microscopy with propidium iodide (sDF-F). The relationship and agreement between the results obtained with both staining methods were analyzed. The values of DNA fragmentation indexes (sDF-F and sDF-B) were compared at each time of chill storage (0, 24, 48, 72, and 96 hours). Additionally, the sperm DNA fragmentation rate (slope) was compared between the methods during chill storage. Good agreement and no significant differences between values obtained with both staining procedures were observed. Finally, the effect of chill storage for up to 96 hours was assessed on sperm motility parameters and DNA fragmentation indexes. Significant differences were found after 48 hours of chill storage, obtaining greater values of fragmented DNA. Progressive sperm motility was lower just after 96 hours of chill storage, and no effect was found in total sperm motility. In conclusion, the Sperm-Halomax kit, developed for canine semen and based on the sperm chromatin dispersion test, can be used accurately under bright-field or fluorescence microscopy to assess the sperm DNA integrity of canine semen during chill storage. The sperm DNA fragmentation index increased after 48 hours of chill storage, thereby detecting sperm damage earlier than other routine sperm parameters, such as sperm motility.

Identification of sperm subpopulations in canine ejaculates: effects of cold storage and egg yolk concentration.

The aim of this study was to evaluate the effects of cold storage and egg yolk concentration on the distribution of spermatozoa within the different subpopulations. Twenty ejaculates from 4 dogs were collected, diluted in either TRIS buffer containing 20% (TEY20) or 10% centrifuged egg yolk (TEY10) and cooled following a conventional protocol. The kinematic parameters of individual spermatozoa were evaluated in fresh ejaculates and after 24 and 72 h of preservation at 5°C. A multivariate clustering procedure separated 54,261 motile spermatozoa into four subpopulations: Subpopulation 1 consisting of poorly active and non-progressive spermatozoa (19.80%), Subpopulation 2 consisting of slow and low-linear spermatozoa (25.21%), Subpopulation 3 consisting of high speed and progressive spermatozoa (23.88%), and Subpopulation 4 consisting of highly active but non-progressive spermatozoa (31.11%). Although, cold storage had a significant (P<0.05) effect on both the frequency distribution of spermatozoa within subpopulations and the motion characteristics of each subpopulation, the sperm subpopulation structure was perfectly maintained after cold storage. Subpopulations 1 and 2 significantly (P<0.001) decreased during cold storage (Subpopulation 1: 26.6, 16.9 and 18.4%; and Subpopulation 2: 33.6, 21.3 and 24.0%, respectively, for fresh, 24 and 72 h post-cooled), whereas Subpopulations 3 and 4 significantly (P<0.05) increased (Subpopulation 3: 16.7, 27.6 and 24.3%, and Subpopulation 4: 23.1, 34.1 and 33.4%, respectively, for fresh, 24 and 72 h post-cooled). Regarding the relative percentage of spermatozoa within each extender, Subpopulation 3 was more frequently observed in TEY20 after both 24 and 72 h of cold storage. Significant correlations (P<0.05) were found between the proportions of spermatozoa assigned to Subpopulation 3 in the fresh ejaculates and those in stored samples after 24 h (r=0.48498). In conclusion, cold storage significantly modified both the specific parameters and the distribution of spermatozoa within subpopulations. These changes did not affect the general motile sperm structure present in dog, which is conserved during cold storage. The analysis of the changes observed in structures of subpopulations also suggests that the TEY20 provide more effective preservation of dog semen during cold storage.