Abstract
Morphometry, growth performance, genetic variation, reproductive biology and cryopreservation of spermatozoa of critically endangered Tor tor were studied. Thirty eight (38) morphometric measurements and 23 morphometric ratios (%) with total length and head length were measured. Of the morphometric characters examined, all exhibited a significantly positive correlation with total length which indicated the isometric growth in all organs under natural condition. The regression coefficient 'b' of different variable characters (Y) on total length (X) indicated that the rate of growth in terms of total length was highest for weight (b=3.326) and lowest for eye diameter (b=0.489). Ratio of head height to head length, eye diameter to head length, and maximum body depth to total length have been found as the distinguishable characteristics of T. tor compared with T. putitora and T. macrolepis. The meristic counts were found similar to those of previous reports except number of caudal fin rays. Out of twenty two (22) morphometric ratios in respect of total length and head length, six (6) characters were found environmentally controlled which indicated that T. tor of the river Someshwari is disturbed from environmental point of view. Twelve months after rearing higher growth in terms of mean final weight (g), weight gain (g), percent weight gain, and SGR were observed with formulated pelleted feed containing 36.67% protein compared to that of Mega feed containing 28% protein. Microsatellite DNA markers (TPM11, TPM13, Tt1.B01 and Tt1.C10 and Tt2.D01) were used to assess genetic variation among individuals of the species. All the individuals of the population were found polymorphic at all loci which indicate genetic richness of the population. The average number of allele was 9.8 in the population. The highest observed heterozygosity (Ho) was recorded from the locus TPM11 (0.9000) followed by the locus Tt2.D01 (0.7333), locus Tt1.B01 (0.3333), locus Tt1.C10 (0.2000) and TPM13 (0.0667). Applying test for fit to Hardy-Weinberg equilibrium it was found that all the individuals of the population deviated from the equilibrium at all loci. The inbreeding coefficient (Fis) value among the individuals of the population was the highest (0.9194) for locus TPM13 and lowest for locus TPM11 ((-0.0227). The GSI value ranged from 0.04±0.01% to 0.65±0.13% for male and 0.19±0.03% to 3.79±0.58% for female during the study period. The highest GSI value for female (3.79±0.58%) was observed in the month of July and for male (0.65±0.13%) in August. The fecundity of mature female T. tor, determined by gravimetric method, ranged from 57, 996 to 77,644 with the total length of fish varied from 57.32 to 70.00 cm. The average numbers of ova present per g of ovary were 424.40±26.39 and 421.18+4.04 in the months of July and August, respectively. During the sampling period of July and August, the oocyte diameters of minimum (0.70-1.00 mm), median (1.10-1.90 mm), and maximum (2.00-2.40 mm) were measured to determine the oocyte growth. The maximum oocyte diameter was 2.40 mm with the prevalence of 18.33% and 29.67% in the months of July to August, respectively. The gonads with immature stages of oocytes found in the months of March (early and late peri nucleolar stage oocytes) to May (early and late yolk vesicle stages). Both early and late yolk granule stage oocytes appeared in the months of July and August. In male gonads, four stages of spermatogenesis were distinguished namely, spermatogonia, spermatocytes, spermatids, and spermatozoa during the study period, where spermatids were prominent in the month of May and spermatozoa in July to August. Therefore, some aspects of reproductive biology of T. tor such as GSI, fecundity, ova diameter and histological observations of gonadal development predicated that the expected breeding season of T. tor is July to August. Activation of spermatozoa motility was evaluated in 0.1% to 1.0% of NaCl solution and motility decreased with increasing concentration of NaCl. Motility of spermatozoa at 0.4% NaCl solution was more or less stable and seemed completely activated whereas at 1.0% NaCl solution spermatozoa were completely inhibited. During evaluation of cryoprotectant toxicity, the motility decreased with increasing concentration of cryoprotectant (5, 10 and 15%) and 10% DMSO with Alsever's solution was found best at 10 min incubation. Alsever's solution, egg-yolk citrate, urea- egg-yolk and two cryoprotectants, Dimethyl sulfoxide (DMSO) and methanol were used to preserve the spermatozoa. The equilibration and post-thaw motility of the spermatozoa were recorded highest from Alsever's solution with 10% DMSO as 90±3.5% and 80±3.5%, respectively. Cryopreservation of spermatozoa of T. tor has been possible but no breeding trial could be conducted due to lack of ovulated eggs. The present findings could be helpful for further research towards the effort for developing management plan as well as ex-situ conservation of T. tor.
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