Punganur is the world’s shortest (80-100cms) humped cattle (Bos indicus), with long tail and switch touching the ground and is at the risk of extinction. There was a significant difference in the mean scrotal circumference, height and width among the bulls, but the difference between the right and left side testicular measurements was not significant. The overall mean ejaculate volume, mass activity, individual motility and live sperms were highly and significantly different among the bulls. The difference in all pre-freeze and post-thaw semen characteristics between Tris and Biociphos-plus extended semen were significant. Leakage of AKP, ACP and GOT before and after freezing in the semen extended with Biociphos-plus extender were significantly higher than the semen extended with the Tris extender but leakage of GPT was not significant. The difference in follicular development between the group I and II cows was significant. But the difference in the total number of ovulations, embryos and unfertilized ova recovered between Group I and II was not significant. Punganur cattle ear skin pieces and DNA were also cryopreserved.