Post-thaw spermatological characteristics of canine epididymal spermatozoa frozen with Tris based extenders containing trehalose and iodixanol

In this study, it was aimed to determine the effect of different doses of trehalose and iodixanol addition to Tris basic semen diluent on spermatological properties in freezing of canine epididymal spermatozoa and to determine the optimal doses of these substances and to reveal the possible synergistic effect of using both together. In the study, epididymal spermatozoon samples obtained from the testes of 2-4 years old, 15-30 kg body weight, mixed breed street dogs brought to the shelter for sterilisation after bilateral orchiectomy operation were used. The study was carried out in two stages. In the first stage, the optimal doses of trehalose (Tr50 - 50 mM, Tr75 - 75 mM and Tr100 - 100 mM) and iodixanol (Io1% - 1%, Io1.5% - 1.5% and Io2% - 2%) added to the Tris-based diluent were determined (no addition was made to the control group), In the second stage, the effects of optimal doses of trehalose (Tr100) and iodixanol (Io2%) and the combined use of optimal doses (Tr100 and Io2%) on spermatological properties were evaluated (no addition was made to the control group). Epidididymal spermatozoa were collected from the testes of three to four male dogs by washing each epididymis with Tris-based diluent using a 0.5 ml syringe into a petri dish. Total motility, progressive motility and density of the recovered epididymal spermatozoa were determined. These evaluations were performed using the SCA® (Sperm Class Analyser) CASA (Computer Assisted Sperm Analyser) device adjusted according to the standard parameters of canine semen. The collected spermatozoa were kept at room temperature (23-25°C) for 5 minutes, then transferred to tubes and pooled. The samples obtained were ensured to be at least 0.5 ml in volume, motility ≥70% and density ≥200-300×106 spermatozoa/mL. After the spermatozoon density was determined, the semen was diluted gradually at a ratio of 1:3. After cooling, glycerolisation (final glycerol ratio 4%) and equilibration, 0.25 ml straws were first frozen at 110°C and stored at 196°C. Two frozen aliquots were thawed in a 37°C water bath in 30 seconds. After equilibration, thawing, endurance and thermal stress, spermatozoon total motility, progressive motility, kinematic motion parameters were analysed by CASA device, HOS test by phase-contrast microscopy and acrosome and membrane integrity by flow cytometry device. When the spermatological findings of the diluent groups to which different amounts of trehalose were added were compared with the control group, no difference was found in terms of the spermatological properties examined (P>0.05), the LIN value of the group using 50 mM trehalose at the post-thawing stage was significantly higher than the other groups (P<0.05). In the post-thawing flow cytometric analyses, it was determined that the group in which 100 mM trehalose was added was the most superior in terms of the ratios of spermatozoon with intact membrane, live spermatozoon with intact acrosome and dead spermatozoon with damaged acrosome compared to the control group (P<0.05). In the groups containing different ratios of iodixanol, there was no difference between the groups in terms of spermatological properties after equilibration and thawing (P>0.05). In post-thawing flow cytometric analyses, the proportion of viable cells with intact acrosome was significantly higher in the group containing 2% iodixanol (Io2%) compared to the control group (P<0.05). In the groups (Control, OpTr100, Opİo2% and OpTr100+Opİo2%) formed with the combined use of optimal doses, no difference was found between the groups in terms of all spermatological properties examined in the equilibration and post-thawing stages (P>0.05). In terms of post-thawing endurance tests, except for LIN and STR values of the control group after 2 hours of incubation, the other spermatological properties examined were similar to each other. It was determined that the LIN value of the control group was higher than the other groups (P<0.05), but statistically similar to the other groups except OpTr100 (P>0.05). In terms of STR value, it was determined that the control group was statistically superior to OptTr100 and Opİo2% groups (P<0.05). There was no statistical difference between the groups in terms of spermatological properties examined after thermal stress tests performed after thawing (P>0.05). The results showed that freezing and thawing of canine epididymal spermatozoa with Tris basic semen diluent to which 100 mM trehalose was added resulted in a higher proportion of intact acrosomes and plasma membrane integrity. At the same time, the addition of 2% iodixanol to the semen diluent was found to be more successful in terms of acrosome intact cell ratio post-thawing. In vitro fertilization (IVF) and artificial insemination studies are needed to fully demonstrate the fertilisation ability of canine epididymal spermatozoa cryopreserved with Tris base diluent to which iodixanol and trehalose were added.