Agricultural Research Management Information System

Home
Research Summary
Search
About Us
- ARMIS
- Brochure
Contact Us
Report
User Request
Data Input
Help
- Operation Manual
  - PDF
  - Video
- Program Area & Commodity
We have reached 37600 number of research entries at this moment.

- Logout

Research Detail

Home
Research
Detail

Refinement of Cryopreservation Techniques and Setting Up Cryogenic Gene Bank of Two Indigenous Catfishes (heteropneustes Fossilis and Clarias Batrachus) for Quality Seed Production

Prof. Dr. Rafiqul Islam Sarder
Professor
Bangladesh Agricultural University, Mymensingh

Mr. Md. Shahiidul Islam
Scientific Officer
Freshwater Station, Bangladesh Fisheries Research Institute, Mymensingh

D. A. H. M. Kohinoor
Senior Scientific Officer
Freshwater Station, Bangladesh Fisheries Research Institute, Mymensingh

Abstract/ Executive Summary:

Heteropneustes fossilis and Clarias batrachus were reared with supplementary feed consisting of rice bran, mustard oil cake and fish meal at the rate of 50%, 40% and 10%, respectively. Milt sample were collected from male broods maintained in the ponds following the procedure of Viveiros et al. (2000). The fish were anaesthetized and sacrificed by spinal transection. Milt sample was placed in an eppendorf tube on ice. The quality of milt was checked by adding 100-200 ?l of tap water and observed its motility under a microscope. Milt having more than 80% motile sperm was used for preservation. Sperm collected from crushed testes was suspended in a suitable extender and kept on ice until use. Milt was diluted at a ratio of 1:4 for urea egg-yolk and egg-yolk citrate, and 1:9 for Alsever’s solution and Ginzburg Fish Ringer. The cryoprotectants, DMSO (dimethyl sulfoxide), methanol and ethanol were mixed with the milt to make the final concentration of cryoprotectant at 5, 10, 15%. The frozen straws were thawed at room temperature and the post-thaw motility was recorded by eye estimation under the microscope at X10 or X40 magnifications.

Keywords: Refinement, Cryopreservation techniques, Cryogenic gene bank, Catfishes

Location: Mymensingh

Start Date: July 2010

End Date: June 2011

Program Area: Variety and Species

Commodity: Fish

Objectives:

Since catfishes are being subjected to artificial breeding for supplying seeds to farmers, the genetic conservation of broods and its maintenance are the top priorities. Production of seeds in the hatcheries is very much encouraging but the quality related parameters must be addressed properly. The overall purpose of the proposed research project is to conserve the existing gene pool and to improve the quality of seed through cryogenic gene banking. The other specific objectives are: To standardize the developed cryopreservation protocols for H. fossilis and C. batrachus sperm; to improve the protocols for fertilization of eggs using cryopreserved sperm; to determine egg: sperm ratio for sustainable use of sperm; to establish a cryogenic gene bank from where cryopreserved sperm can be supplied to the users; and to disseminate the cryopreservation techniques among hatchery operators.

Methodology:

A series of experiments were conducted to develop and standardize the protocols for cryopreservation of sperm of catfish, Heteropneustes fossilis and Clarias batrachus and to establish cryogenic gene bank. Firstly, the activation of sperm of H. fossilis and C. batrachus mixed with four extenders such as Alsever’s solution, egg-yolk citrate, urea egg-yolk and Ginzburg fish ringer was tested using different concentration of NaCl solution and found that the motility of sperm was decreased with the increase of NaCl concentration. Secondly, toxicity of cryoprotectant to sperm was investigated using 5, 10, 15 and 20% concentrations of cryoprotectant with the incubation time of 5 to 45 min. Thirdly, Alsever’s solution, egg-yolk citrate, urea-egg-yolk and Ginzburg fish ringer and three cryoprotectants, DMSO, methanol and ethanol were used to select suitable extender and cryoprotectant. Urea egg-yolk and egg-yolk citrate along with 10% DMSO produced 64.29±1.71% and 57.86±2.4% post-thaw motility for C. batrachus respectively. For H. fossilis egg-yolk citrate produced 82.50±2.04% post-thaw motility while 80±3.22% post-thaw motility was obtained from Ginzburg fish ringer. In fertilization trials, sperm preserved in Alsever’s solution with either DMSO or methanol fertilized eggs and produced hatchlings. Alsever’s solution with DMSO produced best hatching that was 37.97±2.39% for H. fossilis and 50.29±1.22% for C. batrachus. Alsever’s solution with 10% methanol produced satisfactory results (32.65±2.70% and 41.54±5.54% hatching for H. fossilis and C. batrachus respectively). In cryogenic storage the motility of sperm gradually reduced with the progress of storage time and 75% and 82% motility reduction were recorded from Alsever’s solution plus DMSO and Alsever’s solution plus methanol for H. fossilis, and those of 70% and 75% for C. batrachus respectively. The efficacy of cryogenic stored sperm was tested and satisfactory level of fertilization and hatching were obtained from one month- and two months-old cryofreezed sperm.

Source of Information: Annual Research Program 2010-11. BFRI, Mymensingh.

Article Link (Online Journal):

Funding Source:

1. Government

Budget: Three lskh

Total Budget (Tk.) : Three lskh

Achievement/Findings:

Development of protocols for cryopreservation of sperm of H. fossilis and C. batrachus is an important task as the biodiversity of these species prevails under threat. Protocols for cryopreservation of sperm of the two species have been developed through the research project and successes have been achieved in fertilization and hatching using cryopreserved sperm. Successes have also achieved from cryogenic storage of sperm and its application in artificial insemination. However, further research need to be conducted especially for improvement of fertilization and hatching of eggs using the cryopreserved sperm before delivering a complete protocol to the hatchery operators.

Knowledge Management: Report/Proceedings