Cryopreservation of germ cells from genetically proven animals could be a source of restoration tools from the risk of extinction or disappearance of wanted characteristics. Using frozen semen, the genetic gains of Korean native cattle have been increased greatly for 70 years. The preservation of genetic resources as a form of frozen semen straw has limited availability due to the numbers. To circumvent this weakness of frozen semen, we tested two re-freezing methods with different initial thawing temperatures using frozen Korean proven semen and rare breed semen from albino, black and chikso breeders. It has been known that human sperm could resist to cryo-damages by repeated freeze-thaw cycles, but not for Korean proven bulls number (KPN) or for rare breeds. Total 7 frozen semem from brindled(2), black(1), Korean Albino(2) and KPN(1) bulls were used for our research. After thawing straws under 5°C/2min or 37°C/40sec with low temperature water bath and thermo jug, spermatozoa were re-diluted with triladyl diluents after first thawing and re-frozen. Sperm motilities were compared between animals and treated groups after re-thawing. Mean values of motility and viability of refrozen/thawed sperm for expansion of the number of straws were significantly higher in 5°C than in 37°C (P< < 0.05). However, the activity of viable sperm thawed at 5°C was significantly decreased after first and second thawing. It is estimated that re-freezing of frozen semen from rare Korean native cattle is possible with resistant properties of survived spermatozoa.

Cattle breeds were classified previously into three different haplogroups (Y1 and/or Y2 in Bos taurus and Y3 in B. indicus) based on Y chromosome-specific polymorphisms. In particular, a rapid and unambiguous classification method was reported recently. However, a haplogroup classification of Korean native cattle breeds has not been reported. In this study, 196 animal samples from four Korean native cattle breeds (Hanwoo, Chikso, Heugu, and Jeju black cattle) and six exotic breeds were used to determine the Y chromosome-specific haplogroup classification. We amplified an 81 bp indel region within intron 26 of the USP9Y gene and performed electrophoresis to classify the Y1 and Y2 haplogroups. Moreover, enzyme digestion was carried out with the SspI restriction enzyme to classify the Y2 and Y3 haplogroups. Finally, sequence variation in each haplogroup was confirmed by DNA sequencing. All animals in the four Korean native cattle and two exotic breeds (Charolais and Simmental) belonged to the Y2 haplogroup. Three other exotic breeds (Holstein, Angus, and Hereford) belonged to Y1 haplogroup. Japanese black cattle were divided into both the Y1 and Y2 haplogroups. The Y3 haplogroup corresponding to B. indicus was not found in this study. In conclusion, Korean native cattle breeds originated from B. taurus without introduction from B. indicus. In addition, they showed the same paternal heredity pattern which belonged to only Y2 haplogroup. These results can be used to investigate the origin of Korean native cattle breeds.