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Cryogenic Freezing of Silver Carp Spermatozoa for Conservation of Gene Pool

M. S. Hossain
Department of Fisheries Biology and Genetics, Bangladesh Agricultural University Mymensingh-2202, Bangladesh

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

Abstract/ Executive Summary:

A series of experiments were conducted to develop and standardize the protocols for cryopreservation of sperm of silver carp (Hypophthalmicthys molitrix) with the view to conserve gene pool. Three extenders such as Alsever’s solution, urea egg-yolk, egg-yolk citrate, and three cryoprotectants namely DMSO, methanol and ethanol were employed. Cryodiluents were prepared by mixing the cryoprotectants at 10% concentration of the extenders (%v/v). Milt and cryodiluents were mixed at a ratio of 1:9 for Alsever’s solution and 1:4 for urea egg-yolk and egg-yolk citrate solutions. Alsever’s solution, urea egg-yolk and egg-yolk citrate with DMSO found suitable for cryopreservation of sperm and it produced 81 ± 2.24%, 79 ± 5.48% and 74 ± 4.18% post-thawed spermatozoan motility respectively. In optimizing milt dilution, five dilution ratios (1 : 2, 1 : 4, 1 : 6, 1 : 9 and 1 : 12) were tested and among them 1:9 (milt: cryodiluent) demonstrated the highest post-thaw spermatozoan motility with Alsever’s solution (76.67 ± 2.89%) and 1 : 4 produced the same with urea-egg yolk (73.33 ± 2.8%) and egg-yolk citrate (76.67 ± 2.89%). As an optimum cryoprotectant fraction, 10% concentration was found effective to produce the highest spermatozoan motility for all three extenders. Sperm preserved with Alsever’s solution, urea egg-yolk and egg-yolk citrate plus DMSO demonstrated the ability to fertilize eggs and produced hatchlings.

Keywords: Silver carp, Cryopreservation, Sperm, Fertilization, Hatching

Location: Genetics Laboratory, Department of Fisheries Biology and Genetics, Bangladesh Agricultural University, Mymensingh

Start Date:

End Date:

Program Area: Conservation and Biodiversity

Commodity: Carp fish

Objectives:

To cryopreserve sperm of silver carp by developing and standardizing cryopreservation protocols through optimizing cryodiluents, milt dilution and cryoprotectant concentration.

Methodology:

The experiment was conducted in the Genetics laboratory, mini hatchery and wet laboratory of the Department of Fisheries Biology and Genetics, Bangladesh Agricultural University by using the following methods. The brood fish weighing about 1.5 - 3.5 kg were collected. Milt was collected from mature males after being induced with pituitary gland (PG) extract. Sperm samples containing more than 80% motility were used. For preservation of milt, three extenders, Alsever’s solution, urea-egg-yolk and egg-yolk citrate and three cryoprotectant methanol, DMSO (Dimethyl sulphoxide) and ethanol were used. Cryodiluents were prepared by adding 10% cryoprotectant to 90% extender (% v/v). Milt and cryodiluents were mixed at a ratio of 1 : 9 for Alsever’s solution and 1:4 for urea egg-yolk and egg-yolk citrate solutions.Milt sample was first cooled from the ambient temperature (0^oC) to -4^oC at a falling rate of 4^oC per min and then from -4^oC to -80^oC at a falling rate of 10^oC per min. After reaching to -80^oC, samples were removed from the cryochamber and loaded into the canisters and finally placed into the liquid nitrogen (-196^oC) for long-term preservation. For determining the suitable dilution ratio between milt and cryodiluents, milt was diluted with each of the cryodiluents at five different ratios such as 1 : 2, 1 : 4, 1 : 6, 1 : 9 and 1 : 12. Similarly, five different cryoprotectant concentrations such as 5, 10, 15, 20 and 25% were tested for determining its suitable concentration. Fertilization trial was conducted using sperm preserved with Alsever’s solution, urea egg yolk and egg yolkcitrate with DMSO. Eggs were collected from hormone induced (PG plus HCG) female brood by stripping into a plastic bowl immediately after ovulation. Stripped eggs were divided into four batches where each batch contained approximately 1200 eggs; three batches of eggs were fertilized with cryopreserved sperm (sperm preserved with three different cryodiluents) and one batch was fertilized with fresh sperm which was considered as control. Each of the three batches of eggs was fertilized with 12 × 230 μl of cryopreserved milt i.e. 12 straws were used for each batch and each straw contained 230 μl diluted milt. For control, adequate amount of fresh milt was used. After insemination, 5 - 10 ml of 0.9% NaCl solution was added to the egg mass-milt and mixed with a feather for about 1 min. The fertilized eggs were then transferred to incubators for hatching. The equilibrium and post-thawed motility of sperm were analyzed using two-factor randomized complete block design through computer software package (MSTAT). The fertilization and hatching rates were analyzed using one-way ANOVA followed by Duncan’s Multiple Range Test (DMRT).

Source of Information: Progress. Agric. 20(1 & 2) : 99 – 106, 2009, ISSN 1017-8139

Article Link (Online Journal): http://www.banglajol.info/index.php/PA/article/view/16861/11836

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

Fertilization rates of eggs with cryopreserved and fresh sperm were significantly (P<0.01) different but no significant (P>0.05) differences in the hatching rates between fresh and frozen spermatozoa were observed. Fertilization and hatching of eggs using cryopreserved sperm was possible but the hatching rates need to be increased through optimizing techniques of thawing of frozen sperm and fertilization.

Knowledge Management: Journal