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Sperm Cryopreservation of an Endangered Freshwater Spiny Eel, Mastacembelus Armatus (lacepede, 1800) for Biodiversity Conservation in Bangladesh

Mostafa Ali Reza Hossain
Department of Fisheries, Biology and Genetics,Bangladesh Agricultural University, Mymensingh 2202, Bangladesh

Md. Akhtar Hossain
Department of Fisheries, University of Rajshahi,Rajshahi-6205, Bangladesh

Shirin Akter
Department of Fisheries, Biology and Genetics,Bangladesh Agricultural University, Mymensingh 2202, Bangladesh

Mst. Afroza Sultana
Department of Fisheries, Biology and Genetics,Bangladesh Agricultural University, Mymensingh 2202, Bangladesh

Md. Mahbubul Hassan
Department of Fisheries, Biology and Genetics,Hajee Mohammad Danesh Science and Technology University, Dinajpur 5200, Bangladesh

Md. Nahiduzzaman
Department of Fisheries, Biology and Genetics,Bangladesh Agricultural University, Mymensingh 2202, Bangladesh

Abstract/ Executive Summary:

Cryopreservation as a technique has long been used successfully in human, livestock and more recently in fishes. Cryogenic storage (-196 °C, the temperature of liquid N₂) of sperm of an endangered, and popular freshwater fish of Bangladesh - spiny eel, Mastacembelus armatus (Lacepede, 1800) was attempted for ex-situ conservation. Sperm were collected in 0.9% NaCl solution (287 mOsmol kg^-1) from artificially induced males and activated with 0.2% NaCl (67 mOsmol kg^-1) for motility analysis. Motility of the freshly collected sperm was 85% and retained the capacity of forward movement for 170s. The one-step freezing (from 5 °C to -80 °C at 10 °C/min) was carried out in a computer controlled freezer (FREEZE CONTROL® CL-3300; Australia) to cryopreserve the sperm. The highest motility (%) of the post freeze sperm were 52.22 ± 5.27 (mean ± SD) when equilibrated with 10% dimethyl sulfoxide (DMSO) and 48.89 ± 8.66 (mean ± SD) with 10% methanol. The result suggests that sperm of can be cryopreserved in 0.9% NaCl after 10 min equilibration in the cryoprotectants with one step freezing, and can be useful for long term preservation of gamete for revival of endangered M. armatus.

Keywords: Cryopreservation, Endangered spiny eel, Mastacembelus armatus, Sperm, Cryoprotectant

Location: Faculty of Fisheries, Bangladesh Agricultural University

Start Date: 00-00-2009

End Date: 00-00-2010

Program Area: Conservation and Biodiversity

Commodity: Eel

Objectives:

The present study focuses on the state of the art of development of cryopreservation protocol for M. armatus and its implications in conservation strategies.

Methodology:

M. aramatus were collected from the river Brahmaputra adjacent to Bangladesh Agricultural University and cultured in the mini hatchery of the Faculty of Fisheries. They were fed with live Tubifex at 5% bw twice daily. During June-July 2009, the male fishes were injected with pituitary gland (PG) supernatant at 5 mg/kg and released in the conditioning tank for twelve hrs. Gentle pressure was applied on the abdomen, and the sperm was collected in eppendorf tube (1.5 ml) containing 0.9% NaCl solution prepared at 287 mOsmol kg^-1. The samples were placed on ice (4 °C) and brought to the Laboratory of Fish Biodiversity and Conservation, Bangladesh Agricultural University for motility analysis and cryopreservation. A simple solution, 0.9% unbuffered salt (sodium chloride) has been used as extender and produced good sperm survival percentage. With appropriate testing, extenders can be prepared in large batches and stored in refrigerator for few days. Dilution of sperm in extender improves sperm survival duration after storage, and gives a greater volume of the solution thus makes it easier to handle the sperm. Motility of the fresh sperm was evaluated under light microscope (Novex K-range, Holland) at x 400 magnifications (Figure 4). Motility of the collected sperm samples were estimated after activation with 10 μl 0.2% NaCl (67 mOsmol kg^-1) as activating medium with 1 μl of the diluted sperm on a glass slide. The sperm cell showed active forward movement was considered motile, and samples containing more than 80% motile sperm considered for further study. Typical cryopreservation of sperm cells involves the use of chemicals called cryoprotectants, which protects sperm cell from damage during freezing and thawing. Cryoprotectants are classified as intracellular or extracellular depending on whether they penetrate the cell or remain outside of the cell. Two cryoprotectants, DMSO and methanol used in the study at concentrations ranging from 5 to 20% in the solutions prepared for cryopreservation. Toxicity measurement was done evaluating motility (%) of the equilibrated sperm with aforementioned cryoprotectant at different time intervals like 5, 10, 15 and 20 min. Based on the better motility (%) of the sperm, suitable equilibration time has been selected for different concentrations of cryoprotectants for cryogenic storage. For cryopreservation, pre-labeled 0.25-ml French plastic straws (Tiefenbach, Germany) were filled with 0.23 ml of diluted spermatozoa and sealed, and the straws were transferred to a programmable controlled-rate freezer (FREEZE CONTROL® CL-3300; Australia) by computer-based software (CryoGenesisTM V5). The choice of optimal cooling rate is one of the focal points for sperm cryopreservation. In this study, the samples were cooled by one-step freezing (5 °C to -80 °C at a rate of 10 °C). After freezing straws were retrieved from the cryochamber and immediately plunged into the liquid nitrogen (-196 °C) of the cryocan for long term storage. Cryopreserved sperm was transported to the hatchery for fertilization of the eggs. Post freeze motility of the cryopreserved sperm was studied after thawing at 50 °C in water bath for 10 sce after activation of the sperm with 0.2% NaCl.

Source of Information: ISESCO Journal of Science and Technology - Volume 7, Number 12 (November 2011) (57-66)

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

Considering the fragile biodiversity status and high consumer demand of M. armatus, it is not unlikely that the fish might completely disappear from the natural waterbodies if proper steps are not taken immediately. To our knowledge, this is the first attempt to cryopreserve the sperm of the fish successfully, as sperm viability remained unchanged after storage in liquid nitrogen. The post-freeze motility (%) of the cryopreserved sperm is still poor, and it is inevitable to do further research on it. Moreover, the ability to fertilize eggs with cryopreserved sperm is more appropriate criterion to assess the success of the protocol and that has yet to be evaluated. Whatsoever, the experiment was successful in unearthing the appropriate media for dilution and activation of sperm, equilibration time for toxicity tolerance and suitable cooling rate, which would be immensely helpful in further research on exsitu conservation of M. armatus.

Knowledge Management: Journal