Cryopreservation of the Endangered Mahseer (Tor khudree) Spermatozoa: Effect of Dimethyl Sulfoxide, Freezing, Activating Media, and Cryostorage on Post-Thaw Spermatozoa Motility and Fertility

Abstract

The present study was conducted with the goal of evaluating the viability of spermatozoa of the Deccan mahseer (Tor khudree, Cyprinidae) cryopreserved using different strategies. Immotile spermatozoa pooled from 2-4 males were diluted with modified fish Ringer's solution (pH: 7.48) and protected with dimethyl sulfoxide (Me 2 SO) at 5%, 10%, and 15% and subjected to different equilibration periods. Diluted samples (1:10) were drawn into 500 L plastic straws and frozen at a distance of 5 cm from the level of liquid nitrogen (LN 2) and preserved for 385 days in LN 2. Me 2 SO at 10% resulted in higher post-thaw spermatozoa motility rate (46.7%) than 5% or 15% (up to 33.3%) after 385 days of cryopreservation. Of the different equilibration periods, 20-40 min generally produced higher motility (%) rates than 0, 10, 50, 60, 70, 80, or 90 min. The highest post-thaw motility of spermatozoa was obtained when they were frozen at 2 cm (؊120.3°C) above the level of LN 2 and the optimum freezing rate was found to be 14.5 ؎ 0.1°C /min. Freezing spermatozoa at 1 cm (؊160.4°C) or 3 cm (؊103.0°C) or 4 cm (؊77.9°C) from the level of LN 2 resulted in a significant decrease in post-thaw motility. Among the different activating media tested in the presence of different extenders and cryoprotectants, NaCl (0.3%) was found to be the best, followed by glucose (1%), and tap water. In the presence of different concentrations of Me 2 SO, NaCl (0.3%), glucose (1%), and NaCl plus urea (0.3% ؉ 0.4%) generally produced comparable post-thaw motility percentage and duration at all the concentrations of the cryoprotectant. When the spermatozoa were diluted in the Ringer's solution having pH 7.5 and 7.9, cryoprotected with Me 2 SO at 10% (equilibration period: 30 min) and frozen at 2 cm from LN 2 , the motility rates were 43% and 40%, respectively, after 810 days of cryostorage. High fertilization rate (98%) was obtained for spermatozoa cryopreserved up to 780 days and was close to that of fresh spermatozoa (98.5%). However, the hatching rate and fry survival were marginally lower in the treatment than that of control. After 1 year of rearing in ponds, the growth and survival of fish obtained from cryopreserved and fresh spermatozoa were comparable. The fish produced from cryopreserved spermatozoa were as normal as normally produced fish. The importance of cryopreservation of mahseer spermatozoa vis-à-vis establishment of a frozen gene bank is discussed.

Evaluation of toxicity of cryoprotectant to the sperm of mohashol fish, Tor tor (Hamilton) at various concentrations of the salt solution

“…sperm. Basavaraja et al (2006) [3] also reported that DMSO at 10% resulted in higher post-thaw spermatozoa motility (46.7%) than 5% or 15% (up to 33.3%) after 385 days of cryopreservation for Mahseer (Tor khudree). In cryopreservation of common carp (Cyprinus carpio) sperm best motility was obtained when DMSO, methanol, ethanol and DMA were used at 10% concentration (Sarder et al 2009) [16] .…”

Shielding Endangered Lineages via Strategic Biobanking: Evaluating Extenders and Thaw Rates for Optimizing Viability of Cryopreserved Milt from Golden Mahseer (Tor putitora)

“…Percentage motility was microscopically assessed within 30 secs of sample dilution (1:20 activation) and scored on a 5-point scale from 0 (no motility) to 4 (75-100% progressive motility) (Table 1) (Basavaraja and Hedge, 2004;Basavaraja et al, 2006). Motile lifespan quantified duration of forward movement post-activation.…”

“…Four tris-based extenders were formulated with varied buffering, cryo-protective and membrane-stabilizing agents (Basavaraja and Hedge, 2004;Basavaraja et al, 2006;Martínez-Páramo et al, 2009) (Table 2). A standard cryoprotectant (DMSO: dimethyl sulfoxide) at 10% v/v was supplemented (Basavaraja et al, 2006).…”

“…The unique biochemical traits of each fish species influence its vulnerability to cryoinjury, necessitating the creation of tailored extenders and cryoprotectants for their specific reproductive Shielding Endangered Lineages via Strategic Biobanking: Evaluating Extenders and Thaw Rates for Optimizing Viability of... conditions (Kopeika et al, 2003;Hajirezaee et al, 2010). Despite its threatened status, cryopreservation research on the iconic Golden Mahseer remains limited which constrains conservation options (Basavaraj a and Hedge, 2004;Basavaraja et al, 2006;Hajirezaee et al, 2010;Ponniah et al, 1999; Just two studies have explored freezing protocols for the congeneric Deccan Mahseer (Tor khudree) (Basavaraja and Hedge, 2004;Basavaraja et al, 2006)-but direct extrapolation to the Golden Mahseer is complicated by interspecific bio-variability (Nynca et al, 2012).…”

The mighty mahseers of the genera Tor, Neolissochilus and Naziritor: A review on resource distribution, biology, ecotourism and conservation

“…A new diploid cell line (TP-1) was developed for the first time from golden mahseer having immense potential in biodiversity conservation (Lakra et al, 2006). It was observed that the cryopreservation of spermatozoa of Deccan mahseer (T. khudree) using different techniques increases the viability of spermatozoa (Basavaraja and Hegde, 2004;Basavaraja et al, 2006). The phylogenetic relationship among the five Indian mahseer species was done using a ribosomal DNA sequence (45S rDNA) which provided greater understanding of the taxonomic status (Kumar et al, 2015).…”