Sperm cryopreservation of an endangered freshwater spiny eel, Mastacembelus armatus (Lacepede, 1800) for biodiversity conservation in Bangladesh

8 Nahiduzzaman … 1/11/11 14:37 Page 57 ISESCO JOURNAL of Science and Technology Vo l u m e 7 - N u m b e r 1 2 - N o v e m b e r 2 0 11 ( 5 7 - 6 6 ) C Abstract Sperm cryopreservation of an endangered freshwater spiny eel, Mastacembelus armatus (Lacepede, 1800) for biodiversity conservation in Bangladesh -80 °C at 10 °C/min) was carried out in a computercontrolled freezer (FREEZE CONTROL® CL-3300; Australia) to cryopreserve the sperm. The highest motility (%) of the postfreeze 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 longterm preservation of gamete for revival of endangered M. armatus. ryopreservation as a technique has long been used successfully in human, livestock and more Mostafa Ali Reza Hossain*, recently in fishes. CryogeMd. Nahiduzzaman, Md. Mahbubul nic storage (-196 °C, the Hassan1, Mst. Afroza Sultana, temperature of liquid N2) of Shirin Akter and Md. Akhtar Hossain2 sperm of an endangered, and popular freshwater fish *Department of Fisheries, Biology and Genetics, of Bangladesh - spiny eel, Bangladesh Agricultural University, Mymensingh Mastacembelus armatus 2202, Bangladesh (Lacepede, 1800) was 1 Department of Fisheries, Biology and Genetics, attempted for ex-situ conHajee Mohammad Danesh Science and Technoservation. Sperm were collected in 0.9% NaCl solulogy University, Dinajpur 5200, Bangladesh 2 tion (287 mOsmol kg-1) from Department of Fisheries, University of Rajshahi, artificially induced males Rajshahi-6205, Bangladesh and activated with 0.2% *Tel: +880-1711-045364; NaCl (67 mOsmol kg-1) for Email: marhossain@yahoo.com motility analysis. Motility of the freshly collected Keywords: Cryopreservation, Endangered spiny eel, sperm was 85% and retained the capacity of forward Mastacembelus armatus, Sperm, Cryoprotectant movement for 170s. The one-step freezing (from 5 °C to 1. Introduction siltation, anthropogenic activities such as dam construction (mainly for flood control, irrigation and drainage), and unregulated construction of polders (natural depressions enclosed by embankments), hydroelectricity generation, and construction of road networks have been major causes of freshwater species loss. In addition, freshwater resources are subject to severe competition among multiple human stakeholders such as crop farming, aquaculture, and industrial usage. The major threats to freshwater biodiversity in Bangladesh are overefishing, water abstraction for irrigation in to crop land, pollution, massive destruction of fish habitat, and invasion of exotic fish species. Rapid extraction of fish seedstock (for aquaculture) as well as broodfish (for seed production and consumption) from natural rivers and floodplain combined with destructive and unregulated fishing practices (e.g., use of destructive traps, piscicides, monofilament gillnets, and complete dewatering of waterbodies) has threatened a number of valuable native species. Loss of aquatic habitat due to Recently there has been expanded development of cryogenic sperm banks for fish in Europe and North America. These sperm banks are more cost effective 57 8 Nahiduzzaman … 1/11/11 9:05 Page 58 Hossain, Nahiduzzaman, Hassan, Sultana, … / ISESCO Journal of Science and Technology - Volume 7, Number 12 (November 2011) (57-66) TABLE 1. Cryopreservation of sperm of some fish species in Bangladesh than maintaining live gene banks which require dedicated facilities, labor and high costs. Cryogenic gene banking avoids the risk of genetic contamination and requires little space and minimal facilities. Fish sperm cryopreservation assists conservation of fish biodiversity through gene banks of endangered species, and assists aquaculture by providing flexibility in spawning of females and selective breeding through synchronizing artificial reproduction, efficient utilization of semen, and maintaining the genetic variability of broodstocks [Lahnsteiner, 2004]. The technique also ensures preservation of genetic materials of the genetically superior wild fish populations and the gene transfer between wild and hatchery stocks [Tiersch et al. 1998]. Fish group Fish Catla catla Cirrhinus mrigala Indigenous Labeo rohita Labeo calbasu Puntius sarana Cyprinus carpio Hypophthalmichthys molitrix Exotic fishes The sperm cryopreservation protocols for different fish species seem variable and species-specific. Although fish are the main protein source in Bangladesh and other countries in the sub-continent, and the fish biodiversity and production from open water are declining, little attention has been paid to cryopreservation of fish sperm. In India, protocols have been developed with varying success only for a few aquacultured and endangered species [Ponniah 1998]. The trials have mainly concentrated on development of extenders, activation media, dilution rates, activation periods, and sperm-to-egg ratios among species. Hypophthalmichthys nobilis Barbonymus gonionotus Oreochromis niloticus is native to the riverine fauna in the Indian sub-continent and parts of Southeast Asian countries [ITIS, 2011] (Figure 1). This species is a popular food fish that fetches a high market value in its country of origin. M. armatus is a large elongated fish that has a snake-like body without pelvic fins and can reach up to 91 cm in its natural habitat but does not usually exceed 51 cm in captivity. It's anal and dorsal fins are elongated and are connected to the caudal fin and the dorsal fin is preceded by numerous spines. Owing to its gradual disappearance from the natural waterbodies for over exploitation and a number of ecological changes in its natural habitats, the popular fish has been enlisted as an endangered fish in Bangladesh [IUCN, 2000]. Recent study by the Fish Museum and Biodiversity Center (FMBC) of Bangladesh Agricultural University reveals that the extent of occurrence of this indigenous fish in the natural waterbodies has decreased all the more, and recategorized as critically endangered needing immediate actions for conservation [Hossain and Wahab, 2010]. Cryopreservation allows indefinite storage of biological material into liquid nitrogen (-196 °C) without any major change of the biological importance over a time scale of several thousands of years [Baulney et al., 1999]. The method can aid in the conservation actions through preservation of germplasm of rare fishes as well as the more widely used commercial species. In Bangladesh, cryopreservation of fish spermatozoa is relatively new compared to livestock semen although the technique has immense applied value, and it can be an important tool when programs will be designed to produce genetically improved strains of superior fish or repository of the threatened fishes. Cryopreservation protocols have been developed in Bangladesh for some native as well as exotic fish species [Hossain et. al., 2010] (Table 1). So far a few of the threatened species of Bangladesh have been considered for cryopreservation and a lot of actions need to be taken to get the momentum. Cryopreservation of sperm is a useful technique to preserve genetic material of endangered fish species and can be utilized for fertilization in well-designed breeding programs. During protocol development for fish sperm cryopreservation, key parameters of sperm samples (e.g., ionic composition, osmolality) as well as development of appropriate activation media, immobilization solutions, cryoprotective agents, equilibration time, cooling rates, The freshwater spiny eel, Mastacembelus armatus (Lacepede, 1800) belongs to the family Mastacembelidae 58 8 Nahiduzzaman … 1/11/11 9:05 Page 59 Hossain, Nahiduzzaman, Hassan, Sultana, … / ISESCO Journal of Science and Technology - Volume 7, Number 12 (November 2011) (57-66) Figure 1. Freshwater spiny eel, Mastacembelus armatus (Lacepede, 1800). and thawing rates should be given consideration because of differences within and among species. The most widely used cryoprotectant, dimethyl sulfoxide (DMSO), permeates the cells quickly and is usually used at the concentrations of 5-12 %. It has been found to be effective for a number of fish species. Methanol is also a permeating cryoprotectant, known for low toxicity and has been reported to be used successfully at concentrations of 5-20 % for the cryopreservation of several fish species. Usually, after equilibration in cyroprotectant, the diluted sperm is frozen (-80 °C) in straws, with different cooling rates (5-15 °C/min) in the vapor of liquid nitrogen and stored (-196 °C). Later, the samples are thawed at 4-37 °C and used for fertilization of eggs. Sperm cryopreservation protocols are now available for over 200 species of finfish and shellfish around the world. over, breeding success has not been adequately addressed in those studies. Genetic stock conservation for wild and domesticated fishes is very important, as the genetic diversity of every species develops through a long evolutionary process over millions of years. Cryogenic techniques can assist in the conservation of biodiversity, to bring back the threatened species to natural environment with restocking programmes, as well as can improve aquaculture production. The present study focuses on the state of the art of development of cryopreservation protocol for M. armatus and its implications in conservation strategies. 2. Materials and Methods 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 (Figure 2). Fish are the main protein source in Bangladesh and other countries in the sub-continent, and the fish biodiversity and production from open water are declining. In Bangladesh, research on fish sperm cryopreservation was started in early 2004 and protocols are available for a few of the commercially reared species. Unfortunately, very few studies on sperm cryopreservation have been carried out for imperiled fish species in Bangladesh with the vision to conserve their germplasm and genetic resources. Cryopreservation research in Bangladesh has concentrated on selection of suitable combinations of extenders and cryoprotectants, optimal dilution ratios of milt, and optimal cryoprotectant concentrations. More- 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 (Figure 3). 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. 59 8 Nahiduzzaman … 1/11/11 9:05 Page 60 Hossain, Nahiduzzaman, Hassan, Sultana, … / ISESCO Journal of Science and Technology - Volume 7, Number 12 (November 2011) (57-66) Figure 2. The male M. armatus was injected with pituitary gland (PG) supernatant. Figure 3. Collection of M. armatus sperm in 0.9% NaCl solution. 60 8 Nahiduzzaman … 1/11/11 9:05 Page 61 Hossain, Nahiduzzaman, Hassan, Sultana, … / ISESCO Journal of Science and Technology - Volume 7, Number 12 (November 2011) (57-66) 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. 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. 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) (Figure 5). 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. 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 (Figure 6). Postfreeze 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. 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 Figure 4. Sperm motility observation of M. armatus under a microscope. 61 8 Nahiduzzaman … 1/11/11 9:05 Page 62 Hossain, Nahiduzzaman, Hassan, Sultana, … / ISESCO Journal of Science and Technology - Volume 7, Number 12 (November 2011) (57-66) Figure 5. A controlled-rate freezer (FREEZE CONTROL®CL-3300; Australia) and a microscope used in the experiment. Figure 6. Transportation of frozen fish sperm using a cryocan. 62 8 Nahiduzzaman … 1/11/11 9:05 Page 63 Hossain, Nahiduzzaman, Hassan, Sultana, … / ISESCO Journal of Science and Technology - Volume 7, Number 12 (November 2011) (57-66) 3. Results and Discussion 2004] and Razorback sucker, Xyrauchen texanus (70 s) [Tiersch et. al., 1997]. Motility of the freshly collected M. armatus sperm was 85% and retained the capacity of forward movement for 170s after activation with 0.2% NaCl. Sperm motility duration varies according to fish species and retains the capacity of active forward movement from few seconds to several minutes after activation in a hypo-osmotic solution [Alavi and Cosson 2006]. In our study, M. armatus sperm retained motility up to 170s which was shorter compared to the sperm of Muskellunge, Exox masquinongy (6-7 min) [Lin and Dabrowski, 1996], Paddlefish, Polyodon spathula (4-5 min) [Mims, 1991], and longer compared to Olive barb, Puntius sarana (35 s) [Nahiduzzaman et al., 2011], Walleye, Sander vitreus (51 s) [Satterfield and Flickinger, 1995], Colorado Pikeminnow, Ptychocheilus lucius (57 s) [Tiersch et. al., Toxicity of cryoprotectant was evaluated with DMSO and methanol at concentrations 5% to 20% with subsequent times form 5 to 20 min each. The motility of fresh sperm before equilibration with cryoprotectants was 80%. Sperm suspended in 5% DMSO showed no significant motility (%) change over 20 min, and with an increase in DMSO concentration (10% or more) sperm motility decreased after 10 min equilibration (Figure 7a). The sperm motility (%) pattern was similar when equilibrated with methanol at aforementioned concentrations and durations (Figure 7b). At the end of toxicity experiment, M. armatus sperm was cryopreserved after 15 min equilibration with 5% cyroprotectant and 10 min equilibration with rest of the concentrations (10, 15 and 20%). 63 8 Nahiduzzaman … 1/11/11 9:05 Page 64 Hossain, Nahiduzzaman, Hassan, Sultana, … / ISESCO Journal of Science and Technology - Volume 7, Number 12 (November 2011) (57-66) Figure 7. The toxicity of two different cryoprotectants, DMSO (top) and methanol (bottom) at final concentrations of 5%, 10%, 15% and 20% when equilibrated with sperm from Mastacembelus armatus in 0.9% NaCl solution at an osmolality of 287 mOsmol kg-1. Each point represents the mean ± SD of three males. methanol showed significantly better post-freeze motility (%). In summary, M. armatus sperm could be cryopreserved in 0.9% NaCl solution having 10% DMSO or 10% methanol (10 min equilibration) with a cooling rate of 10 °C/min from 5 °C to -80 °C and finally plunged into liquid nitrogen. Cryoprotectants are essential for cryopreservation of sperm to protect against intracellular ice crystal formation during freezing and thawing [Yang et. al., 2007]. Suitability of cryoprotectants, their concentration and gamete contact time vary among fish species [Vuthiphandchai, 2009]. The difference is related with the cryoprotectant permeability, extent of toxicity [Cabrita et. al., 2003], and sperm survival depends on delicate balance between toxicity and capacity to protect gamete [Lahnsteiner, 1996]. In toxicity study of M. armatus sperm, 5% DMSO and 5% methanol were found nontoxic after 20 min exposure. 10% DMSO and 10% methanol were found nontoxic up to10 min and consecutive exposure killed a significant portion of the gamete. Post-freeze sperm motility is one of the most important indicators of suitability of cryopreservation [Akcay et. al., 2004]. After cryopreservation of M. armatus, marked differences in sperm motility was observed with 5% DMSO or methanol, which confirmed that the concentration was not toxic, but failed to protect sperm during freezing and thawing. Similar results were also reported after cryopreservation of the sperm of olive barb (Puntius sarana) [Nahidizzaman et. al., 2011] and red snapper (Lutjanus argentimaculatus) [Vuthiphandchai et. al., 2009]. When cryopreserved with 10% DMSO or methanol after 10 min equilibration, highest percentage of motile sperm was found and gave the impression that it was the best of the combinations tested. This is an After cryopreservation, sperm motility (%) reduced significantly compared to pre-freeze motility at every concentrations and equilibration time (Table 2). Pre-freeze and Post-freeze motility were significantly affected by cryoprotectant concentrations. Among the concentrations evaluated, sperm suspended in 10% DMSO or 10% 64 8 Nahiduzzaman … 1/11/11 9:05 Page 65 Hossain, Nahiduzzaman, Hassan, Sultana, … / ISESCO Journal of Science and Technology - Volume 7, Number 12 (November 2011) (57-66) TABLE 2. Sperm motility percentage (mean ± SD; minimum-maximum) of M. armatus (N = 9) before and after cryopreservation suspended in different concentrations of DMSO and methanol. The sperm samples were cooled at 10 °C/min from 5 °C to -80 °C (equilibration time 15 min for 5% DMSO and methanol, and 10 min for 10%, 15% and 20% DMSO and methanol) Cryoprotectant DMSO Methanol Concentration Pre-freeze motility Post-freeze motility 5% 66.67±5.0a (60-70) 33.33±5.0c (30-40) 10% 65.56±4.41a (60-70) 52.22±5.27a (50-60) 15% 56.67±0cd (50-60) 40.0±5.0b (40-40) 20% 53.33±5.0d (50-60) 36.67±5.0bc (30-40) 5% 62.22±5.27bc (50-70) 25.56±8.33d (20-30) 10% 63.33±6.01b (50-70) 48.89±8.66a (40-60) 15% 46.67±5.27e (40-50) 25.56±5.0d (20-30) 20% 43.33±0f (40-50) 20±5.0e (20-20) sperm equilibrated with 15% and 20% DMSO or agreement with sperm cryopreservation protocol developed for olive barb (Puntius sarana) [Nahidizzaman et. al., 2011], yellowfin sea bream, Acanthopagrus latus [Gwo, 1994] and yellow perch (Perca flavescens) [Ciereszko et. al., 1993]. In this study, cryopreservation of methanol resulted lower percentage of sperm motility. This suggests that the concentrations were toxic and unsuitable for cryopreservation of M. armatus sperm. Conclusions 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. Acknowledgments The authors would like to acknowledge the funding assistance from COMSTECH and ISESCO through the “Ex-situ conservation of three indigenous fishes of Bangladesh using cryogenic gene bank” project. References (Silurus glanis) testicular spermatozoa after freezing with different cryoprotectants. Cryobiology, 39, 177-184. [1] Akcay, E., Bozkurt, Y.S.S.E., and Tekun, N., 2004, Cryopreservation of Mirror Carp Semen. Turkish Journal of Veterinary and Veterinary Animal Science, 28, 837-843. [2] Alavi, S.M.H., and Cosson, J., 2006, Sperm motility in fishes. (II) Effects of ions and osmolality: a review. Cell Biology International, 30, 1-14. 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