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Cryogenic Preservation of Critically Endangered Cirrhinus Reba Fish Sperm

Shirin Sultana
National Institute of Biotechnology, Ganakbari, Ashulia, Savar, Dhaka, Bangladesh

Md. Mahmud Hasan
National Institute of Biotechnology, Ganakbari, Ashulia, Savar, Dhaka, Bangladesh

Mohammad Shahdat Hossain
National Institute of Biotechnology, Ganakbari, Ashulia, Savar, Dhaka, Bangladesh

Md. Rashidul Islam
Hajee Mohammad Danesh Science & Technology University, Dinajpur, 5200

Md. Salimullah
National Institute of Biotechnology, Ganakbari, Ashulia, Savar, Dhaka, Bangladesh

Md. Rafiqul Islam Sarder
Bangladesh Agricultural University, Mymensingh, Bangladesh

Md. Saidul Islam
National Institute of Biotechnology, Ganakbari, Ashulia, Savar, Dhaka, Bangladesh

Md. Moniruzzaman
Ministry of Science & Technology, Peoples Republic of Bangladesh

Jahangir Alam
National Institute of Biotechnology, Ganakbari, Ashulia, Savar, Dhaka, Bangladesh

Abstract/ Executive Summary:

The natural population of Cirrhinus reba in Bangladesh has been decreasing rapidly and is subsequently categorized as threatened. The study was carried out to develop a suitable protocol for C. reba sperm cryopreservation. The sperm activation was observed in 0.1%-1.1% NaCl solution with highest motility 92.5 ± 2.5 (mean±SE) at 0.3% NaCl solution and lowest (zero motility) at 1.1%. Two different extender such as inorganic chemical Alsever’s solution, organic egg-yolk citrate and two cryoprotectants namely methanol and dimethylsulfoxide (DMSO) were used in toxicity analysis at 5%, 10% and 15% concentration. The cryopreservation was carried out with three diluents (Alsever’s solution plus 10% DMSO, Alsever’s solution plus 10% methanol; Egg-yolk citrate plus 5% DMSO) in two steps freezing protocol (from room temperature to -4°C at a rate of 4°C per min and then from -4°C to -80°C at a rate of 10°C per min). Alsever’s solution with 10% DMSO showed best performance and producing 85±5.77 and 58.75±3.15% equilibration and post-thaw motility, respectively. This protocol will be helpful to conserve the gene pool of C. reba.

Keywords: Diluents, Freezing protocol, Cirrhinus reba

Location: Mymensingh district

Start Date: 00-04-2014

End Date:

Program Area: Animal Health and Management

Commodity: Fish

Objectives:

Considering the potentiality of cryopreservation techniques in aquaculture, research on sperm cryopreservation protocol of different freshwater species has been developed in Bangladesh. Cryopreservation of sperm of different threatened fishes like sarpunti , veda, pabda, baim have also been done. However, the aim of this research was to develop a suitable cryopreservation protocol for the critically endangered C. reba fish sperm.

Methodology:

2.1 Brood collection and rearing The fishes were collected from natural sources of Mymensingh district in April 2014. Mature male (milt or sperm comes after gentle pressure on abdomen) and female (swollen abdomen) of C. reba fishes were reared in the ponds of Fisheries Faculty of Bangladesh Agricultural University, Mymensingh, Bangladesh. The fishes were fed twice daily by commercial feed.

2.2 Sperm collection and quantifying sperm motility The brood males were collected from the pond and acclimatized in the tank of hatchery area after measuring the length (cm) and weight (kg). The broods were injected with pituitary gland extract (dry PG was collected from the local market). After six hours of injection (2 mg kg-1) milt was collected in an eppendorf tube by gentle pressure on abdomen. Sperm concentration was determined in triplicate counts by use of haemacytometer. The milt was diluted 1000 times in distilled water. 10-12µl of the diluted sample was taken on haemacytometer and covered with a cover slip (Marienfeld, Germany) and expressed as the number of cells x109 ml-1. After measuring the concentration, the motility of sperm in each tube was checked out under the microscope taking about one-two µl of milt on a glass slide with 100-200 µl distilled water.

2.3 Evaluation of sperm and cryopreservation Sperm samples were exposed to 10 graded dilutions of NaCl solutions. Sperm motility was observed in 0.1 to 1.1% NaCl solutions for activation. This experiment was done to formulate an extender which osmolality should be resembled to the milt and sperm will remain inactive in it during cryopreservation. To determine a suitable diluent for cryopreservation of sperm of C. reba, two extender Alsever’s solution, egg-yolk citrate and two cryoprotectants namely methanol and DMSO were used in cryopreservation trials. Cryoprotectant concentrations were maintained according to the percentage (5, 10 and 15%) in the final suspension. Collected milt samples were diluted with the diluents at 1:4 (milt: diluent) for egg-yolk citrate and 1:9 for Alsever’s solution. After observing the equilibration motility, the diluted milt was taken into 250µl plastic straws and sealed the open end. Then the straws were placed into the cryochamber and initiated cooling. Computer controlled freezer (CL-3300) was used to freeze the sample using two steps freezing protocol, where milt sample was firstly cooled from ambient temperature (25°C) to -4°C at a rate of 4°C per min and then from -4 to -80°C at a rate of 10°C per min. Finally the frozen straws were placed into the liquid nitrogen (-196°C) for long term preservation. Straws were retrieved from liquid nitrogen container and thawed at room temperature for 30-40 seconds to assess the motility under microscope.

2.4 Statistical Analysis Data of motility for activation, toxicity and equilibration expressed in percentage were converted to arcsine transformation. Data of activation and toxicity were analyzed with one factor ANOVA of SPSS v 20 and the means were separated by Least Significant Difference (LSD) at 5% level of significance.

Source of Information: International Journal of Fisheries and Aquatic Studies 2017; 5(5): 334-339

Article Link (Online Journal):

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

This study was conducted to develop a cryopreservation protocol for Cirrhinusrebafish sperm. The findings from this experiment are that the Alsever’s solution and 10% DMSO will be suitable diluent (extender + cryoprotectant) for this fish sperm with a cooling rate of 0^o C to -80^o C at a decreasing rate of 10^o C/min. This protocol will be helpful to conserve the gene pool of C. reba. However, further research needs to be performed for standardizing thawing and fertilization techniques, suitable egg-sperm ratio etc.

Knowledge Management: Journal