A study was conducted to standardize a protocol for cryopreservation of spermatozoa of the endangered mahseer, Tor khudree (Sykes) and T. putitora (Hamilton). The suitability of the cryoprotectants, dimethyl sulphoxide (DMSO) and glycerol, and the combination of the two were tested. Four equilibration periods and four freezing rates were also tested for their standardization. A combination of 9% DMSO and 11% glycerol gave signi¢cantly higher mean percentage of hatching in both T. khudree (45.59 AE 1.86%) (control 71.08 AE 0.59%) and T. putitora (45.00 AE 1.25%) (control 73.48 AE 1.19%) among the eight di¡erent treatments. Among the four di¡erent equilibration periods tested, the equilibration period of 30 min À1 yielded the highest mean hatching percentage in T. khudree (39.46 AE 1.94%) (control 71.70 AE 0.61%) and T. putitora (38.28 AE 1.06%) (control 73.11 AE 0.82%). Freezing straws at a height of 8 cm above LN 2 surface for 10 min À1 gave higher hatching percentages for both T. khudree (41.75 AE 1.72%) (control 73.99 AE 1.17%) and T. putitora (41.34 AE 2.04%) (control 72.48 AE 1.51%). The study reports the superior performance of the combination of DMSO and glycerol for the ¢rst time.

Studies on the physico-chemical characteristics of seminal plasma, along with ultrastructure and mitochondrial activity of fresh spermatozoa of the endangered Deccan mahseer Tor khudree were undertaken. The ultrastructure and mitochondrial activity of fresh spermatozoa were compared with those of cryopreserved-thawed spermatozoa to understand the nature and extent of cryo-damage. Physico-chemical analyses of fresh milt revealed sperm density of 3.93±0.11 x 10 7 spermatozoa ml -1 , spermatocrit value of 67.08±1.22%, higher K + concentration of 13.16±0.121 mg l -1 , total reducing sugar and total protein concentration of 47.31±0.82 and 19.60±0.66 mg 100 ml -1 respectively. Ultrastructure of the fresh spermatozoa by both scanning (SEM) and transmission electron microscopy (TEM) revealed the spherical head without any acrosomal complex, small mid piece with mitochondria and a long tail. Cross section of tail by TEM revealed typical 9+2 doublet arrangement of the axoneme. Head measured 1.86±0.006 µ in dia with a mid-piece length of 0.53±0.012 µ and tail length of 33.53±0.220 µ. Ultrastructural damages to the spermatozoa following cryopreservation included, loosening of chromatin and disruption of the cytoplasmic membrane as compared to that of fresh spermatozoa. Nitroblue tetrazolium (NBT) assay revealed low levels of activity of the enzymes of the mitochondrial complex in cryopreserved-thawed spermatozoa when compared to fresh spermatozoa indicating damage to the functional integrity of the enzymes of the mitochondrial enzyme complex.