67 Indian J. Fish., 63(1) : 67-73, 2016 Studies on fresh milt parameters and cellular changes during cryopreservation of spermatozoa of Deccan mahseer Tor khudree (Sykes, 1839) RAVINDRAGOUDA PATIL * , WAZIR SINGH LAKRA, SHRINIVAS JAHAGEERDAR GOPAL KRISHNA AND ASIM KUMAR PAL Fish Genetics and Biotechnology Division, Central Institute of Fisheries Education, Versova, Mumbai - 400 061 Maharashtra, India * Fisheries Research and Information Center, Karnataka Veterinary, Animal and Fisheries Sciences University Hessaraghatta, Bengaluru - 585 401, Karnataka, India e-mail: ravi.patil30@gmail.com ABSTRACT 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. Keywords: Cryopreservation, NBT assay, Physico-chemical characteristics, Spermatozoa, Tor khudree, Ultrastructure Introduction Physico-chemical characteristics of fsh milt determine the fertilisation ability of spermatozoa (Rurangwa et al., 2004). Fish seminal plasma contains various organic and inorganic components which support the viability of spermatozoa (Hajirezaee et al., 2010). Hence, biochemical analysis of seminal plasma plays a signifcant role in the assessment of milt quality (Billard et al., 1995), which has applications related to artifcial fertilisation and sperm preservation (Billard, 1978). Also, studies on the ultrastructure of fsh spermatozoa are very much useful in understanding the phylogenetic relationships among different species (Jamieson, 1991) and they also help in comprehending damages following cryopreservation (Calvi et al., 1994; Lahnsteiner et al., 1996). Deccan mahseer, Tor khudree belonging to the family Cyprinidae, once formed a major fshery from the rivers and streams of Western Ghats and today, it is enlisted as an endangered species (Ogale, 1994; Basavaraja and Keshavanath, 2000; Dahanukar et al., 2004). Attempts for its ex situ conservation using cryopreservation of spermatozoa have been made (Basavaraja et al., 2002; Patil and Lakra, 2005). For effcient mass production of T. khudree fngerlings for ranching purposes and also for development of effcient cryopreservation methodology, it is necessary to study the physico-chemical characteristics of seminal plasma. The present study was undertaken with the objectives of investigating the physico-chemical characteristics of seminal plasma as well as for studying the ultrastructure and mitochondrial activity of fresh spermatozoa in comparison with that of cryopreserved- thawed spermatozoa to understand the nature and extent of cryo-damage. Materials and methods The present study was undertaken in the mahseer farm and hatchery complex of the Tata Power Company, Lonavala, Maharashtra State, bordering Sahyadri range of the Western Ghats. DOI: 10.21077/ijf.2016.63.1.52060-09