The successful cryopreservation of spermatozoa of the beagle dog for AI is essential for the establishment of the genetic banks of drug detection dogs. The beagle dog is widely used for drug testing and chosen for breeding by breeders. However, the use of cryopreserved beagle semen is limited by the lower number of offspring of dog species. In this study, 3 highly trained beagle dogs were chosen and their semen was cryopreserved for the next generation. The effects of dilution methods of beagle semen were tested using a direct dilution method at RT and a 2-step dilution method at 5°C. As a control group, the effects of a direct dilution method of semen on the percentage of motile sperm and progressive motility were analysed by computer-assisted semen analysis system (SAIS, Korea), and abnormality of spermatozoa was examined by Diff Quik staining. A total of 9 samples from 3 dogs were extended in 4% glycerol containing Tris-egg yolk diluents at approximately 22 to 25°C. The diluted semen was cooled to 5°C within 2h. The packed 0.5-mL straws were placed 5cm above the surface of LN for 10min and then plunged in. A 2-step dilution method was conducted using the same procedures of freezing, but the first dilution was done with glycerol-free diluent. After cooling to 5°C within 2h, the second diluent with 8% glycerol was added to the same volume of diluted semen at 5°C and stabilised for 1h. After thawing for 45s at 37°C, the semen from the 2-step dilution method showed the higher percentage of motile sperm (65.4±6% v. 45.3±8%; P<0.05) and progressive motility (41.6±5.3% v. 32.3±3.7%; P<0.05). However, the abnormalities between groups showed no differences. The results suggest that the optimal method for freezing beagle dog spermatozoa is a 2-step dilution process that consists of the first dilution at RT and the second dilution with glycerol at 5°C into diluted semen.
Semen from Korean Native Black roosters (Ogye) was cryopreserved with 8% N-Methylacetamide (MA) in HS-1 diluent, of the following composition (per 100 mL): glucose 0.2 g, trehalose dihydrate 3.8 g, l-glutamic acid monosodium salt 1.2 g, potassium acetate 0.3 g, magnesium acetate tetrahydrate 0.08 g, potassium citrate monohydrate 0.05 g, BES 0.4 g, Bis-Tris 0.4 g, and gentamicin sulfate 0.001 g. Ogye semen was collected 2 times a week by dorsal-abdominal massage and cooled into ice slurry. During dilution, semen was diluted by 2 steps. First, HS-1 diluent that was supplemented with respective concentration of MitoTEMPOL, mitochondria-specific antioxidant was added into fresh semen with same volume. Second, after waiting for 10 min, 16% MA containing diluent was added into first diluted semen so the final concentrations of MA were adjusted to 8%. The concentration of the MitoTEMPOL of first diluent was adjusted to 0.1, 1, 5, and 10 μM. After freezing for 30 min by exposure to liquid nitrogen vapor 4 cm above the surface of liquid nitrogen, the semen was thawed 5°C for 2 min 1 to 3 weeks later. The viability and longevity of thawed Ogye semen were analysed by sperm movement methods. Briefly, 5 μL of thawed semen was placed onto a Makler chamber and the movement of spermatozoa was recorded for 10 s by digital camera and saved as movie files on the computer. With digital rewinding for 1 s of saved movies, the non-motile and motile sperm was counted using a manual counter. The resulting numbers of respective spermatozoa were analysed by Student t-test. The 0.1 and 1 μM treated semen showed significant increases in viability compared with control, 5 μM and 10 μM MitoTempol (77.3 and 84.2% v. 65.4, 53.2, and 21.5%; P < 0.05). The longevity of frozen/thawed Ogye semen for 3 h was also higher in 0.1 and 1 μM treated groups than control, 5 μM, and 10 μM (67.5 and 54.2% v. 36, 5.2, and 1.2%; P < 0.05). With these results, utilisation of mitochondria-specific antioxidant for freezing of Ogye spermatozoa could increase the viability and longevity of frozen-thawed semen. However, treatment with concentrations >1 μM showed negative effects on freezing of Ogye chicken semen. These findings could be helpful for cryobanking of rooster semen for preservation of selected breeders from malignant viral avian disease.
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