Abstract
This study was aimed at the phenotypic and genetic characterization of an endangered carp, kalibaus Labeo calbasu and development of sperm cryopreservation protocol for ex-situ conservation. Landmark-based morphometrics and meristic counts were applied to evaluate the population status of L. calbasu collected from four wild (the Jamuna, the Padma, the Halda and the Haor) and one hatchery sources. Six (standard length, head length, eye length, peduncle length, pre-orbital length and maxillary barbel length) of 12 morphometric measurements, two (pectoral fin rays and scales above the lateral line) of 9 meristic counts, and all (except- 4 to 5 and 5 to 7) of 22 truss network measurements revealed a highly significant inter stock variation (P<0.01, P<0.001) between all samples. Plotting Discriminant function DF1 and DF2 showed a clear differentiation between stocks for both morphometric and landmark measurements. For both morphometric and landmark measurements, the first and second DF accounted 73% and 15.7% of among group variability and together they explained 88.7% of the total group variability. For the morphometric and truss network measurements, plotting DFs revealed high isolation of the stocks. The dendrogram based on morphometric and truss distance data placed the Jamuna and Hatchery in one cluster. The Halda and the Padma constructed one cluster with one sub-cluster with Haor population. Microsatellite DNA marker analysis was also carried out to assess the population genetic structure of L. calbasu, collected from four wild and a Hatchery sources. Twenty five (25) primers developed from rohu (Labeo rohita), catla (Gibelion catla) and carpio (Cyprinus carpio) were tested for PCR amplification of which nine (9) primers were amplified successfully. Out of 9 (nine) loci, four microsatellite markers (Lr10, Lr21, Lr24 and Lr26) developed from rohu and one microsatellite marker (CcatGI) developed from catla were analyzed to test the genetic variability of the kalibaus stocks. The maximum number of alleles observed in loci Lr10, Lr21, Lr24 and Lr26 and CcatG1 were 4, 6, 4, 5 and 10, respectively. The loci were found to be polymorphic (<P95) in all the populations. The average number of allele was highest in the Jamuna population (5.6) followed by the Padma (5.2), the Halda (5) and the Haor (5) populations. The Hatchery populations possessed the least number (4.8) of average alleles. Therefore, wild populations possessed the highest average allele number than the cultured stock. The observed average heterozygosity (H.) in the Jamuna population (0.770) was the highest followed by the Halda (0.667), the Haor (0.661) and the Padma (0.642) populations. Except loci Lr10 and Lr24 in the Halda and locus Lr10 in the Padma and Hatchery populations, significant deviations from Hardy-Weinberg Equilibrium (HWE) were detected in all cases. The FST values and the Nm values indicated high level of differentiation and a low level of gene flow between the populations. The largest genetic distance value (D = 0.548) was measured between the Jamuna and the Hatchery population while the smallest value (D =0.124) was observed between the Padma and the Halda populations. A simple protocol of refrigeration storage and cryopreservation of endangered kalibaus sperm was developed for artificial fertilization and long term preservation. Sperm was collected in Alsever's solution (296 mOsmol kg') and activated with distilled water (DW) (24 mOsmol kg') to characterize motility. Maximum motility (90%) was observed within 15 s after activation with DW, and sperm remained motile for 60 s. Sperm activation was evaluated at different osmolalities of NaCl solution and motility was completely ceased when osmolality of the extender was ≥ 287 mOsmol kg'. Of the two diluents (Alsever's solution and 0.9% NaCl) used for refrigeration storage, sperm motility in Alsever's solution was found to be better at 1:10 and 1:15 ratios. Cryoprotectant toxicity of sperm was evaluated with 5, 10 and 15% dimethyl sulfoxide (DMSO) or methanol. After equilibration at different times, 15% DMSO was found to be highly toxic and excluded for cryopreservation. The one-step freezing protocol (from 5°C to -80°C) with two cooling rates (reducing at 5°C and 10 °C/min) was carried out in a computer-controlled freezer (FREEZE CONTROL® CL-3300; Australia) in which 10°C/min cooling gave significantly (P = 0.011) better post-thaw motility. After fertilization with fresh eggs, significant differences were observed (P = 0.001) between cryopreserved and fresh sperm as well as different egg to sperm ratios (P = 0.001). When fertilized with cryopreserved sperm, 1: (4.13×105) egg to sperm ratio gave better results and thought to be optimal for kalibaus. Cryopreserved sperm can facilitate hatchery operations and for long-term conservation of genetic resources to contribute in the recovery of endangered fish. Mass seed production and conservation of the best stock through proper management is highly recommended to save the endangered and valuable kalibaus form extinction.