Niranjan Sarangi

Recent advances in carp seed production and milt cryopreservation

The fish-seed production industry in India has recorded remarkable growth over the last three decades. The hypophysation technique was successfully introduced into India in 1957 and steady progress towards the refinement of the technique has been registered, which has revolutionized carp seed production in the Indian subcontinent. Advancement of carp maturity through brood stock management and multiple breeding has enabled spawn production well ahead of the monsoon and even beyond, ensuring seed availability throughout the year. The quality of seed is an important consideration for commercial aquaculturists. So, partial stock replenishment in carp hatcheries is practised to overcome the problem of inbreeding, which otherwise leads to poor growth of carps. Similarly, gametes of improved stock are cryopreserved and utilized for quality seed production as well as upgrading the brood stock of carps. The gamete cryopreservation protocol for carps is the focus of this review. An attempt has also been made to incorporate information on carp brood stock management, inducing agents, and seed production, including hatchery management.

Development of 21 new microsatellite markers in Labeo rohita (rohu)

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Use of the Non-Toxic Cryoprotectant Trehalose Enhances Recovery and Function of Fish Embryonic Stem Cells Following Cryogenic Storage

Fish embryonic stem (ES) cells derived from of blastulae (64 cell stage embryo) of Labeo rohita were propagated in culture and retained their ES cell-like properties after cryogenic storage (-196 degrees C, i.e., liquid nitrogen). Toxic effect of DMSO (dimethyl sulphoxide) on stem cells during preservation process has been reported to restrict therapeutic applications. In this study we reduced the concentration of DMSO and added the non-toxic cryoprotective agent (CPA) trehalose. Cryopreservation of ES cell colonies was done at 5, 25 and 52 passages with 0.2 M trehalose and 0.8 M (DMSO). A combination of both the cryoprotective agents (non-toxic and toxic) demonstrated better survival and recovery of ES cells than the DMSO used alone. Use of this CPA combination in the freezing media gave an optimum viability of more than 83 % in a slow freezing protocol. Trehalose showed a definite advantage over DMSO in terms of viability and intactness of ES cell colonies with evenly distributed morphology. There was no significant difference observed in the expression levels of cell surface markers like stage specific embryonic antigen-1 (SSEA-1) and alkaline phosphatase (ALP) between early and late passages after 60 days of post-thawing. More than 90 % of the ES cell colonies showed extensive expression of ALP and positive expression of SSEA-1 from an early stage of ES cells culture up to passage 52 (in our study) in the presence of leukemia inhibitory factor (LIF) and without feeder cells. Further, thawed ES cells showed a normal karyotype and maintained an undifferentiated state through out the study. This study on ES cell cryopreservation and subsequent retention of stem cell properties without feeder cells using a non-toxic cryoprotectant trehalose would be highly useful for future in vitro differentiation, manipulation of fish ES cells and as a model for mammalian ES cell culture.

Bioaccumulation of sodium, potassium, calcium and magnesium in Rohu, Labeo rohita (Ham.) fry.

The bioaccumulation of alkali and alkaline earth metals such as Na, K, Ca and Mg were studied in the fry of rohu, Labeo rohita (Ham.), a species of freshwater aquaculture importance in India. The experiments were conducted in laboratory conditions exposing the rohu eggs up to fry stage in one experiment; and fry to advanced fry stage in other experiment, to different concentrations of the salts. Sodium chloride (0.15, 1.5, 15, and 150 mg/l), potassium chloride (0.015, 0.15, and 1.5 mg/l), magnesium chloride (0.15, 1.5, 15, and 150 mg/l), and calcium chloride (0.15, 1.5, 15, and 150 mg/l) were used as test salts. The tests were conducted in 20-litre capacity glass aquaria stocked with 800 eggs up to fry stage; and in the second experiment with 60 numbers of fry up to advanced fry stage. Control animals were reared in the laboratory without addition of salts. The experiments were conducted in triplicate and each one continued for one-month duration. Normal rearing practices were followed and 50 per cent test solutions from each aquarium were replaced with fresh ones in every week. The bioaccumulations in wet weight basis in whole tissue of fry reared from eggs and in advanced fry exposed to respective salts at the end of the experiment were 100-572 and 91-147 ppm for sodium; 71- 104 and 13-22 ppm for potassium; 16-32 and 24-48 ppm for calcium; and 210-660 and 150-300 ppm for magnesium respectively. In control the bioaccumulations for fry reared from eggs, and in advanced fry were 150-212 and 371-412 ppm for sodium; 85-99 and 80-85 ppm for potassium; 18-22 and 5.5-7.8 ppm for calcium; and 250-260 and 15 ppm for magnesium respectively. No definite relationship could be established between the alkali and alkaline earth metals concentration in aquatic environment and bioaccumulation in fish tissue, except sodium and calcium in fry stage.