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Development of a Sperm Cryopreservation Protocol of Gangetic Mystus (mystus Cavasius)

MM Islam
Department of Fish Biology and Biotechnology, Chittagong Veterinary and Animal Sciences University, Khulshi, Chittagong 4225

MN Noor
Department of Fisheries Biology and Genetics, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh

AA Islam
Department of Fisheries Biology and Genetics, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh

MRI Sarder
Department of Fisheries Biology and Genetics, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh

MZ Islam
Department of Fisheries Biology and Genetics, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh

DA Jahan
Bangladesh Fisheries Research Institute, Mymensing 2202, Bangladesh

Abstract/ Executive Summary:

Cryopreservation is considered as one of the most useful techniques for long-term preservation of genetic material specially sperm of fish. This study focused on the development of a sperm cryopreservation protocol for indigenous near threatened gulsha (Mystus cavasius) and a number of experiments were conducted for the purpose. To collect milt, male gulsha were sacrificed and milt was suspended in extenders. Different concentrations of NaCl were used to evaluate the activation of sperm motility and it decreased as the concentration of the extending media increased, therefore, motility was completely inhibited at 0.8% and 1.2% NaCl solution when sperm suspended in Kurokura-2 and Alsever’s solution, respectively. The toxicity of cryoprotectants to sperm were evaluated using two cryoprotectants, dimethyl sulfoxide (DMSO) and methanol along with the extenders, Alsever’s solution and Kurokura-2 solution. DMSO and methanol with 5% and 10% concentrations produced significantly higher motility during 5 and 10 min incubation and their 15% concentration found toxic to sperm. Alsever’s solution with 10% DMSO produced best equilibration (83.75±2.39%) as well as post-thaw motility (67.5±3.23%) while Kurokura-2 solution with DMSO produced similar equilibration motility (81.25±2.39%) but the post-thaw motility (50.0±6.12%) was significantly much lower than that of Alsever’s solution. Sperm preserved with Alsever’s solution plus DMSO produced highest fertilization, 72.5±7.5% and hatching, 56.8±5.6% while fresh sperm yielded 85.0±5.0% and 74.8±3.6% fertilization and hatching, respectively. The protocols that have been developed can be used for conservation of genetic materials of M. cavasius and other endangered fish species and new generations of them can be propagated using the cryopreserved sperm.

Keywords: Mystus cavasius, Sperm, Cryopreservation, Breeding, Conservation

Location: Faculty of Fisheries, Bangladesh Agricultural University (BAU), Mymensingh, Bangladesh and Bangladesh Fisheries Research Institute, Mymensingh

Start Date:

End Date:

Program Area: Conservation and Biodiversity

Commodity: Aquatic animal

Objectives:

To assess the quality of sperm; toxicity level of cryoprotectants to sperm; suitability of selective diluents and efficacy of cryopreserved sperm in artificial breeding.

Methodology:

Experimental fish collection and rearing- Matured M. cavasius were collected from natural sources (rivers, haors) and stocked in ponds and cisterns of Faculty of Fisheries, Bangladesh Agricultural University (BAU), Mymensingh, Bangladesh and Bangladesh Fisheries Research Institute, Mymensingh. A commercial supplemental feed named Mega feed containing about 35% protein was provided two times a day at 4-5% of total body weight of fish. To increase the natural food production of the pond, organic (cow dung) and inorganic (urea and phosphate) fertilizers were also applied at a rate of 2.0 kg/decimal and 150 g/decimal respectively, at 15 days interval. Liming (250 g/decimal/month) was also done to ensure the quality of water. Selection and conditioning of brood fish- Matured males were selected on the basis of desired phenotypic characteristics. They were caught from the pond 4-6 h prior to hormone treatment and kept in cistern for conditioning. During conditioning, no feed was supplied to them. Additional aeration was ensured by continuous supply of water through PVC pipe over the cistern. Sperm collection, quality assessment and its counting- For sperm collection male brood fish, 6-12 g in body weight were transferred from the rearing pond to the cistern of the mini-hatchery and induced with pituitary gland (PG) hormone. Male broods were induced with a single dose of PG extract of 2 mg /kg body weight. After hormone injection the fish were kept in cistern for about 7 h and the testes were collected by dissection as milt cannot be collected through stripping. The testes were weighed and put in a watch glass placed on ice. As the milt was viscous, a small amount of extender solution (Alsever’s solution and Kurokura-2 solution) was added to the testes separately and crushed them gently using a scissor so that the sperm is suspended in the extender. The sperm suspension was collected using a micropipette and kept in an eppendorf. The eppendorf was placed on ice. Since the quality of sperm can be varied between samples, its motility was observed under a light binocular microscope. To standardize milt dilution and also to estimate the density of sperm per straw, the concentration of milt was determined by counting the sperm using a haemocytometer. The concentration of fresh sperm ranged from 9.65×10⁹ to 1.09×10¹⁰ cells/ml. Estimation of sperm motility About 1- 2μl sperm suspension was put on a glass slide, and 80-100μl of distilled water was mixed to the suspension to activate the sperm. Immediately after activation the motility of sperm was examined under a compound microscope. The sperm which had active movement were counted and expressed as percent motility. For each milt sample, the estimation of sperm motility was done for at least three times in five fields observed each time. Collection of eggs for fertilization Matured females, 20–40g in body weight, were caught from the stocking ponds and transferred to the cistern (216 × 182 × 54 cm; water temperature, 26–28 ºC) for acclimatization for at least 6 h before they were induced with PG extract. Two PG hormone injections were administered at 6 h interval with the doses of 6 mg/kg and 12 mg/kg body weight for the first and second injection respectively. The hormone-injected brood fish were retained in the cistern for ovulation which takes 6–8 h. Experiment-I: Activation of sperm at various concentrations of NaCl Activation of sperm motility was performed at different concentrations of NaCl solution (Sarder et al., 2012). After maceration of testes collected sperm was suspended in extender and placed on ice. A good number of male fish were sacrificed for each extending medium. Twelve graded dilutions of NaCl solution (from 0.1% to 1.2%) were prepared by dissolving NaCl with distilled water. For activation of the sperm 1-2 µl of sperm suspension was taken on a glass slide and 20 µl of NaCl solution from each of the graded dilutions was added to it. Then sperm motility in each of the dilution was examined under microscope. The motility (percentage) and swimming duration (second) of activated sperm at different concentrations of NaCl solution were recorded. These parameters were determined by calculating time difference from activation to the completely immotile or near to immotile condition of the sperm. Statistical analysis Motility of sperm in experiments I, II, III were presented as percentage and all percent motility values were subjected to arcsine transformation before analysis. Data of experiments I and II were analyzed using Independent -samples T test of SPSS v 16 and the means were separated by Least Significant Difference (LSD) at 5% level of significance. To analyze the effects of different extenders and cryoprotectants and their combinations (Expt. III) on both equilibration and post-thaw motility of sperm, one way ANOVA of SPSS (version 16) was used. Means were separated by Duncan’s Multiple Range Test (DMRT) at 5% level of significance. For breeding trial (Expt. IV), Independent-samples T- test of SPSS (version 16) was applied to compare the performance of sperm preserved with different diluents.

Source of Information: Progressive Agriculture 27 (4): 517-529, 2016

Article Link (Online Journal):

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

Considering the threatened state and consumer choice of M. cavasius, it is utmost necessary to take immediate measures to protect their disappearance from natural water bodies. To our knowledge, this is the first attempt of cryopreservation of sperm of the fish as a conservation approach; it will help to conserve the existing gene pool of this endangered species. Though the cryopreservation protocol has been developed by this experiment, the fertilization and hatching rates of eggs should be improved before disseminate the techniques to hatchery operators. In addition, for sustainable use of sperm suitable egg sperm ratio and feasibility of cryogenic gene banking should be investigated.

Knowledge Management: Journal