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Cryopreservation of Spermatozoa of Mrigal, Cirrhinus Cirrhosus with a View to Minimize Inbreeding and Hybridization

M. R. I. Sarder
Department of Fisheries Biology & Genetics, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

S. M. Rafiquzzaman
Freshwater Station, Bangladesh Fisheries Research Institute, Mymensingh-2200, Bangladesh

R. Sultana
Department of Fisheries Biology & Genetics, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

M. Faridul Islam
Department of Fisheries Biology & Genetics, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

Abstract/ Executive Summary:

To develop and standardize the cryopreservation protocol of C. cirrhosus sperm, a series of experiments were conducted. Seven extenders, egg-yolk citrate, urea egg-yolk, 0.9% NaCl, Kurokura-2, Ma and Mb and four cryoprotectants viz. DMSO, glycerol, methanol and ethanol were used to find out the suitable cryodiluent. Among the cryodiluents egg-yolk citrate with DMSO yielded (mean±SD) highest post-thaw motility (83%±5.70) that was closely followed by egg-yolk citrate with methanol (81%±6.51) and urea egg-yolk with DMSO (79%±6.47). Six dilution ratios such as 1: 2, 1: 4, 1: 7, 1: 10, 1: 15, and 1: 20 (milt:cryodiluent) were used to determine the suitable milt dilution and found that sperm motility percentage increased roughly with the increase of dilution ratio. Different dilution ratios exhibited marked differences in the post-thaw spermatozoan motility (P=0.000) and revealed that it has an interaction effect with the extenders (P<0.05) but not with the cryoprotectants (P>0.05). To determine the optimal cryoprotectant concentration, an increasing series of cryoprotectant concentrations from 5 to 30% (v/v) were tested. DMSO at 10% concentration produced averagely highest post-thaw motility with all three extenders. Methanol and ethanol produced best result at 10% concentration but their lower (<7%) and higher (>20%) concentrations decreased spermatozoan motility.

Keywords: Cryopreservation, Spermatozoa, Inbreeding, Hybridization

Location: Fisheries Faculty premises, Bangladesh Agricultural University, Mymensingh

Start Date: 00-00-2009

End Date: 00-00-2009

Program Area: Animal Health and Management

Commodity: Carp fish

Objectives:

1. To develop and standardize the cryopreservation protocol of C. cirrhosus sperm.

Methodology:

Male and female broods were collected from different sources (Halda river-origin and different hatcheries) and stocked in the ponds at the vicinity of Fisheries Faculty premises, Bangladesh Agricultural University, Mymensingh. Brood fish were reared with supplementary feed comprising of mustard oil cake, rice bran, wheat bran and fish meal two times a day at 5% of their total body weight. Vitamin-E was supplemented with the given feed to enhance the gonad development. To develop and standardize the protocols for cryopreservation of Mrigal sperm four experiments were conducted. Determination of suitable extenders and cryoprotectants and their combinations was the first experiment. Finding out the suitable sperm: cryodiluent dilution ratio and concentration of cryoprotectant in the cryodiluent were the second and third experiments. Determine the effects of cryopreservation on fertilization and hatching of eggs was the fourth experiment. To carry out the first experiment the following procedures were maintained. Mature male with desired phenotypic characteristics were selected and induced by injecting PG extract to get sufficient amount of milt. Excess moisture, urine, gut extrudes and mucus were wiped from the area of the genital pore with absorbent paper. Gentle abdominal pressure was applied to collect the milt. Micropipette and tips were used at the time of collection of milt. The collected milt was transferred to eppendorfs or plastic tubes and stored on ice to prevent quality deterioration during further processing. After collection the quality of sperm was checked under microscope by placing 1-2μl diluted sperm on glass slide. Only samples containing more than 80% motile cells by eye-estimation were used for cryopreservation. The number of sperm per ml of milt was estimated using a haemacytometer counter. Counting of sperm is needed to standardize the degree of dilution of milt and to determine the density of sperm per straw for maintaining desired level of egg: sperm ratio during fertilization of eggs. consist of extenders and cryoprotectants. The extender is a solution that contains organic and inorganic chemicals which increase the efficacy of cryopreservation of sperm. Cryoprotectants are mixed with extender to protect the cell from damage during cooling and freezing. Six extenders namely egg-yolk citrate, urea-egg-yolk, Kurokura-2, Ma, Mb, and 0.9% NaCl and four cryoprotectants viz. DMSO, glycerol, methanol and ethanol were used throughout the experiment. Pure milt is not suitable for freezing. So, collected milt was diluted with the cryodiluent (extender + cryoprotectant) at different ratios depending on the type of extender. For egg-yolk citrate, urea-egg yolk, Ma and Mb milt was diluted with the cryodiluent at a ratio of 1:4 (milt: cryodiluent), and for Kurokura-2 and 0.9% NaCl solution, 1:9 dilution ratio was maintained. Cryodiluent was prepared by adding 10% cryoprotectant (e.g. DMSO, glycerol, ethanol and methanol) to 90% extender by volume. The milt and cryodiluent were kept on ice prior to dilution. The diluted milt was equilibrated for 10 min at room temperature and the motility of the equilibrated sperm was checked under microscope before filling straws. Equilibrated milt sample was drawn into the straw and free end of the straw was sealed using heat. The straws were finally placed in the cryochamber and initiated cooling. Computer controlled freezer (CL 300) was used to freeze the samples using the following program. Two-step freezing method was applied where the milt was cooled from +200C to -40C at a rate of 40C per minute, then from -40C to -800C at a rate of 100C per minute and finally transferred into liquid nitrogen. Straws were retrieved from the LN dewar using tweezer and thawed in room temperature. One to two μl of post thawed milt sample placed on to glass slide and activated by adding conditioned water to assess the motility using microscope. To determine the suitable dilution ratio twelve cryodiluents originated from three extenders such as egg-yolk citrate, urea-egg-yolk and 0.9% NaCl and four cryoprotectants namely DMSO, glycerol, methanol and ethanol were tested. Collected milt was diluted with each of the cryodiluent at six different ratios such as 1: 2, 1: 4, 1: 7, 1: 10, 1: 15, and 1: 20. Other procedures such as milt collection, quality assessment, equilibration, freezing and thawing were same as experiment 1. The effect of cryoprotectant concentration was tested using four cryoprotectants (DMSO, glycerol, methanol and ethanol) with each of the three extenders used in the experiment 2. The cryoprotectants were used at 5, 7, 10, 15, 20 and 30% (%v/v) of the extender and thereby, twenty four cryodiluents were generated from each extender. Data were analysed using two-factor and three-factor ANOVA. Significant results (at P < 0.01 and P < 0.05) were further tested by Duncan’s Multiple Range Test (DMRT) to separate means.

Source of Information: J. Bangladesh Agril. Univ. 7(1): 211–218, 2009; ISSN 1810-3030

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

Different dilution ratios exhibited marked differences in the post-thaw spermatozoan motility (P=0.000) and revealed that it has an interaction effect with the extenders (P<0.05) but not with the cryoprotectants (P>0.05). To determine the optimal cryoprotectant concentration, an increasing series of cryoprotectant concentrations from 5 to 30% (v/v) were tested. DMSO at 10% concentration produced averagely highest post-thaw motility with all three extenders. Methanol and ethanol produced best result at 10% concentration but their lower (<7%) and higher (>20%) concentrations decreased spermatozoan motility.

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