Comparative Analysis of One-step and Two-step Dilution on Quality of Frozen Semen in Kintamani Dogs

Preservation of sperm by freezing allows breeding dogs that are separated over long distances. To increase the fertility of frozen and then thawed spermatozoa, they must be able to survive the process. The current study aimed to evaluate the sperm motility and DNA integrity of Kintamani dogs extended in extenders with one-step and two-step dilution techniques. Ejaculates collected from four dogs were used in the current study. The semen was divided into two equal parts and diluted with extenders using two different dilution techniques, namely One-step dilution in Tris egg yolk containing 7% glycerol, and a two-step dilution technique diluted in an initial 2:1 with an extender, containing 20% egg yolk without glycerol. The same volume of the second extender was added, including 14% glycerol. The sample was loaded into 0.25 ml straws, cooled to 4°C for 4 hours, equilibrated, and then plunged into the liquid nitrogen. The sperm motility was evaluated using Computer-Assisted Sperm Analysis (CASA), and DNA integrity was assessed using Acridine Orange (AO) stained. Results showed that the sperm motility of Kintamani dogs in extenders using two-step dilution was significantly higher compared to the one-step dilution technique. In addition, the obtained results indicated that two types of dilution steps in Kintamani dog semen were not detrimental to the sperm DNA integrity during the freezing process. In conclusion, extenders with two types of dilution techniques could maintain sperm motility above 30%, and no difference between one and two steps dilution was detected.