Veterinary session

Recent years have seen a drastic decline in biodiversity. In addition to the traditional in vitro conservation of genetic material, there is a need to establish a well-functioning protocol to cryopreserve the genetic material of a male of an endangered species or with valuable genetics. Storage of epididymal sperm is a feasible method to achieve this goal, however, time of sample delivery and freezing technique are key factors affecting the success rate.

Our aim was to investigate the freezing ability of canine epididymal spermatozoa in fresh stadium and after 24 hours storage at 4°C with two different freezing protocols (ultra-rapid freezing [UR] and vitrification [VF]).

The samples were collected from the tail of the epididymis from 10 mixed-breed dogs. Each sample (fresh and 24-hour storage) was cryopreserved by using the ultra-rapid and the vitrification method. Following parameters were evaluated immediately after harvesting, after 24 hours of cool storage of one testicle at 4°C and post-thawing: total and progressive motility, normal morphology rate, rate of acrosome defects, rate of detached heads, rate of tail defects and proAKAP4 concentration.

Concerning the total motility, no significant difference was found between the UR and fresh groups. There was no significant difference in the progressive motility between the UR 24h group compared to the fresh groups (Fresh 0h and Fresh 24h), but we found significantly lower progressive motility in the UR 0h group compared to both fresh 0h and 24h. However, significantly lower total and progressive motility in VF groups were found compared to both fresh and UR groups. Regarding the morphology analysis, incubation did not have an effect on any of the morphological parameters we examined in any of the freezing groups (p=0.1). In contrast, significant effect of freezing method (p<0.0001) on the rate of cells with normal morphology was found. The rate of normal morphology was significantly lower in the UR group and also in VF group compared to the fresh samples. A significant effect of the freezing method was also observed for acrosome defects (p=0.0137). The rate of sperms showing acrosome defect in UR (p =0.04) and in VF (p=0.01) was significantly higher compared to the fresh samples. In case of detached head and tail defects, in addition to incubation, the freezing method had no effect either. Assessing the proAKAP4 level, higher concentration was found in fresh and VF groups than that of UR group.

In our study, we found that total motility was not affected by incubation for 24 h, but the percentage of progressive motile cells was not significantly reduced in the group incubated for 24 h before freezing compared to fresh frozen samples. These data indicate that when transporting the heritable material of a freshly dead individual to the laboratory, it is recommended to store the epididymis at 4°C for 24 h to maintain better motility.