The effect of storage conditions of the testis-epididymis complex after castration on the quality of canine epididymal spermatozoa before and after cryopreservation.

Abstract

Cryopreservation of canine semen is often used for the export of sperm all over the world, and long term storage of the genetic material of a stud dog or canine species on the verge of extinction. With improvements in assisted reproductive techniques (ART), it is now possible to use cryopreserved epididymal sperm for inseminations. Canine epididymal sperm has shown its fertilizing abilities. Owners of canines of high emotional or genetic value that cannot mate and from whom no ejaculate can be collected could profit from this technique. In case, isolation and freezing of sperm cannot be done on the site of castration, the organs may need to be shipped to a laboratory equipped for this purpose. Therefore, the aim of this study was to compare two storage conditions of the testis-epididymis complex after castration on the quality of canine epididymal spermatozoa before and after cryopreservation. After elective castration in seven dogs (n=7), the organs were either stored for 1-4 hours at room temperature (20-25C; short-term storage) or overnight for 15-20 hours at refrigerator temperature (4-8C; overnight storage). Dimension of the testis were taken using a sliding calliper, weight of the testis and epididymis were taken using a balance. Thereafter, sperm was isolated using the flushing technique and whenever this was not possible the mincing technique was employed. Sperm quality was measured by viability and morphology (anilin-eosin stain), and by Computer Assisted Sperm Analysis (CASA) parameters. There was a significant positive linear correlation between testis weight and testis volume and number of isolated sperm cells (r= 0.787 and r= 0.778 P<0.05). Epididymal weight was not correlated with number of isolated sperm cells. Overnight storage did not have a significant effect on motility, progressive motility and percentage of live cells (P>0.05). Overnight storage had an effect on percentage of bent tails, after overnight storage the percentage of bent tails increased from 4.3% to 34.2% (P<0.05). Processing of semen had a significant effect on percentage of motile cells, this percentage dropped from 72.4% to 18.7 % after freezing and thawing (P<0.05). Processing of semen resulted in a significant decline of percentage progressive motile cells from 45.1 % to 3.0% (P<0.05). The processing of semen had a negative effect on the percentage of live cells, they dropped from 94.5% to 56% after freezing and thawing. Processing of semen also caused a drop in total number normally built and motile cells (TNB), the TNB dropped from 146.5 million cells to 6.8 million cells. Sperm storage overnight at 4 to 8°C is not feasible due to the induction of bent tails. In this study, the loss of motile, normal built sperm cells during the freezing procedure was too large to practically use epididymal sperm for insemination purposes.