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Cryofreezing of Grass Carp (ctenopharyngodon Idella) Spermatozoa for Ex Situ Conservation

M. R. I. Sarder
Department of Fisheries Biology and Genetics, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

M. A. Hossain
Department of Fisheries Biology and Genetics, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

M. S. Hossain
Department of Fisheries Biology and Aquatic Environment, Faculty of Fisheries, Bangabandhu Sheikh Mujibur Rahman Agricultural University (BSMRAU), Salna, Gazipur, Bangladesh

Abstract/ Executive Summary:

The experiment was conducted in the gene tics laboratory, mini hatchery and wet laboratory of the Department of Fisheries Biology and Genetics, Bangladesh Agricultural University, Mymensingh. The methods that have been followed were mentioned below:

Experiments were conducted to develop and standardize the protocols for cryopreservation of sperm of grass carp (Ctenopharyngodon idella). Seven extenders such as Alsever’s solution, Urea egg-yolk, Egg-yolk citrate, Kurokura-1, Kurokura-2, 0.9% NaCl and 0.6% Glucose and five cryoprotectants i.e. DMSO, methanol, ethanol, DMA and glycerol were employed for finding suitable combinations. Cryodiluents were prepared by mixing the cryoprotectants at 10% concentration of the extenders (v/v). Milt and cryodiluents were mixed at a ratio of 1:9 for Alsever’s solution, Kurokura-1, Kurokura-2, 0.9% NaCl and 0.6% glucose, and 1:4 for urea egg-yolk and egg-yolk citrate. Among the 35 combinations of extenders and cryoprotectants, Alsever’s solution with ethanol and methanol, urea egg-yolk and egg-yolk citrate with DMSO found suitable for preservation and it produced 74 ± 2.44%, 72 ± 2.54%, 76 ± 2.44% and 75 ± 2.23% spermatozoan motility at the post-thaw period respectively. Rest of the combinations, on the other hand, produced <60% motility at the post-thaw period and glycerol was found to be clotted after freezing. The dilution of milt with cryodiluent has been tested using six dilution ratios (1:2, 1:4, 1:7, 1:9, 1:15, 1:20) and found that 1:9 dilution ratio produced the highest post-thaw spermatozoan motility with Alsever’s solution (>75%) and 1:4 with urea eggyolk and egg-yolk citrate (>70%). As an optimum level of cryoprotectant, 10% concentration was found effective to produce significantly highest (P<0.05) percentage of spermatozoan motility compared to those of other four concentrations (5, 15, 20 and 30%).

Keywords: Grass carp, Cryopreservation, Sperm, Conservation

Location: Genetics laboratory, mini hatchery and wet laboratory of the Department of Fisheries Biology and Genetics, Bangladesh Agricultural University, Mymensingh.

Start Date:

End Date:

Program Area: Variety and Species

Non-commodity: Carp fish

Objectives:

To conserve the existing gene pool of grass carp, an attempt has been taken to cryopreserve the sperm by developing cryopreservation protocol through optimizing cryodiluents, milt dilution and cryoprotectant concentration.

Methodology:

More than 30 brood fish of grass carp each weighing about 2-4.5 kg were collected from good hatchery sources and stocked in the pond in the vicinity of Fisheries Faculty premises. A supplemental feed comprising of mustard oil cake, wheat bran and rice bran was provided at the rate of 4-5% body weight two times a day. Vitamin- E was supplemented to the feed for enhancing gonadal development of fish. Besides, green grass and leaves of banana were also provided to the fish. Inorganic and organic fertilizers were applied regularly to increase the natural food (phytoplankton and zooplankton) production. Milt was collected from matured males after being induced with pituitary gland (PG) extract. Pure and concentrated white-coloured milt was collected in an eppendorf tube by applying gentle abdominal pressure and kept on ice. The quality of milt samples were checked under the binocular light microscope by placing 1-2 μl of milt on a glass slide with about 100-200 μl tap water. Milt samples containing more than 80% motility were considered as study sample. Similar to the second experiment, three extenders and five cryoprotectants were used to determine the suitable cryoprotectant concentration in the third experiment. Five different concentrations such as 5%, 10%, 15%, 20% and 30% were tested. Procedures for collection, preparation and cooling of sperm used for the first experiment were remained same in the second and third experiments. The equilibrium and post-thawed motility of spermatozoa were analyzed using twofactor randomized complete block design through computer software package (MSTAT).

Source of Information: Progress. Agric. 21(1 & 2): 141–150, 2010 ISSN 1017-8139

Article Link (Online Journal): http://www.banglajol.info/index.php/PA/article/view/16763/11793

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

In breeding trials, sperm preserved with the cryodiluents did fertilize eggs but could not produce hatchlings. The reason for failure of hatching of eggs was not clear but the consequences of the improper thawing technique of cryopreserved spermatozoa prior to fertilization or any unwanted error in fertilization process could be responsible. Cryopreservation protocol for grass carp spermatozoa has been developed and standardized through this study but further research need to be conducted especially on improvement on fertilization and hatching of eggs with cryopreserved sperm before demonstrating full protocol to the hatchery operators. The development of such protocols for cryopreservation of sperm may facilitate sustainability in aquaculture and the establishment of a national facility for gene banking of fish gametes and DNA material.

Knowledge Management: Journal