Sessions

During our research, we examined the freezability of sperm collected from the epididymis of male dogs. In recent years, epididymal sperm has received increasing attention because it can be used as a last resort to preserve the genetic material of valuable breeding animals. Using this technique provides access to the genetic material of animals that die unexpectedly.

Between May and September 2025, we processed epididymides from 14 dogs at the Laboratory of Andrology and Assisted Reproduction. Data from 10 of these individuals could be used for our experiment.

The samples of the dogs were from the University of Veterinary Medicine Obstetrics Clinic neutering program. The age of the males was between 8 months and 5 years and their weight ranged between 1,9 and 27 kg.

During the experiment, the effectiveness of two different freezing media was compared: Steridyl (Minitube), registered for use in cattle, and CaniRep Uppsala Equex II (CaniRep Hb), registered for use in dogs. We collected the sperm cells from the epididymis using a modified SESA (single incision epididymal sperm aspiration) technique. Then we added the sperm to 1 ml phosphate-buffer saline. After that, we used CASA software (MICROPTIC S.L. SCA®) to assess cell motility and prepared smears for morphological evaluation (Spermac stain) and for DNA analysis (acridine orange stain). The samples were divided into two equal parts and for cryopreservation we used the two different cryopreservative media. The samples were placed into 0,25mm straws and stored in liquid nitrogen. The samples were thawed later using two different methods (with/without thawing medium) and subjected to the same analyses as the fresh samples.

The statistical analyses were performed with the help of the R statistical programme. During the examination of the fresh samples, the mean progressive motility was 26,88±4,97%, the mean concentration was 268,95±163,07 x106/ml, and the mean total cell count was 268,5±163,84 x106.The proportion of sperm with normal morphology was 72%±11,39% acrosome integrity was 96,7±4.74%, and DNA integrity had a mean value of 99,2±0,79%. The morphological averages of smears prepared after thawing did not differ significantly from those observed in the fresh samples with respect to normal morphology, DNA integrity, or acrosome integrity (p > 0.05).

The progressive motility after thawing also did not show significant difference between the fresh and the frozen samples. However, a significant difference (p=0.045) was observed in total motility between the fresh sample and the sample preserved with Steridyl, thawed without thawing medium (87,47±6,97% vs. 71,37±12,43%). Meanwhile, when thawing medium was added, the difference compared to the fresh sample was no longer significant (87,47±6,97% vs.73,79±16,06%).

Overall, the results indicate that Steridyl freezing medium, provided similarly good outcomes to CaniRep Uppsala Equex II when used to cryopreserve epididymal sperm from dogs.