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Cryogenic Freezing of Silver Barb (barbonymus Gonionotus) Spermatozoa for Gene Pool Conservation

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

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

M. S. Hossain
Lecturer
Department of Fisheries Biology and Genetics, Sheikh Fajilatunnesa Mujib Fisheries College, Melandh, Jamalpur, Bangladesh

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

Abstract/ Executive Summary:

Experiments were conducted to develop and standardize the protocols of cryopreservation of sperm of Barbonymus gonionotus. Three extenders Alsever’s solution, urea-egg-yolk and egg-yolk citrate and four cryoprotectant methanol, ethanol, DMSO and DMA 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. Alsever’s solution with 10% DMSO showed the best performance and produced 78 ± 2.55% sperm motility at the post-thaw period. Urea egg-yolk and egg-yolk citrate with 10% DMSO produced 76 ± 1.87% and 74 ± 1.87% post-thaw motility respectively. When cryopreserved sperm was stored in liquid nitrogen for 130 days, a gradual reduction in motility ranging from 31.25 to 37.50% was observed. This could be happened due to frequent opening of the nitrogen dewar that could cause thawing of the sample. In breeding trials, sperm preserved with Alsever’s solution and urea egg-yolk plus 10% DMSO produced 14.28 ± 7.06% and 15.46 ± 5.50% hatching respectively. In contrast, sperm preserved with egg-yolk citrate and 10% DMSO produced poor hatching as 8.01 ± 2.15%.

Keywords: Silver barb, Cryopreservation, Sperm, Fertilization

Location: Fisheries Faculty premises, Bangladesh Agricultural University, Mymensingh.

Start Date:

End Date:

Program Area: Conservation and Biodiversity

Commodity: Fish

Objectives:

To develop the cryopreservation protocols by optimizing cryodiluents and by assessing storage effect on post-thaw motility of sperm and fertilization capability of cryopreserved sperm.

Methodology:

The broods of silver barb were collected from Bangladesh Fisheries Research Institute (BFRI) and from some other known hatcheries and stocked in the ponds at the vicinity of Fisheries Faculty premises, Bangladesh Agricultural University, Mymensingh. Fish were reared with supplementary feed comprising of mustard oil cake, rice bran and wheat bran at the rate of 45%, 30% and 25%, respectively. Vitamin E was provided with the feed to enhance the gonadal development. The feed was supplied twice a day at the rate of 4- 5% of the total body weight of the fish. Three experiments were conducted in which determination of the suitable extender, cryoprotectant and their combination was the first one. Assessment of storage effect on post-thaw motility of sperm and fertilization capability of cryopreserved sperm were the second and third experiments. Though the three experiments were conducted separately the procedures for collection of milt and its preparation for preservation were same in all the experiments. In the first experiment, three extenders and four cryoprotectants were used. The extenders were Alsever’s solution, urea egg-yolk and egg-yolk citrate while methanol, ethanol, DMSO and DMA were the cryoprotectants. Cryodiluents were prepared by mixing 90% extender with 10% cryoprotectant (v/v). Milt samples were diluted with the cryodiluents at the ratio of 1 : 4 (milt : cryodiluent) for egg-yolk citrate and urea egg-yolk and 1 : 9 for Alsever’s solution.Data were analyzed by two-factor randomized block design using computer software package (MSTAT) and one way ANOVA followed by Duncan Multiple Range Test (DMRT).

Source of Information: Progress. Agric. 20(1 & 2) : 117 – 124, 2009, ISSN 1017-8139

Article Link (Online Journal): http://www.banglajol.info/index.php/PA/article/view/16863/11839

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

Three extenders Alsever’s solution, urea egg-yolk and egg-yolk citrate and four cryoprotectant viz. methanol, ethanol, DMSO and DMA were used to preserve the sperm. The effects of these extenders and cryoprotectant on the motility of sperm at both equilibrium and post- thaw periods. most of the cryodiluents produced >80% equilibrium motility but it was significantly reduced after thawing. The dilution ratio between milt and cryodiluent was selected as 1:4 for egg-yolk citrate, urea egg-yolk, and 1:9 for Alsever’s solution as these dilutions were found. Among the four cryoprotectant used DMSO performed best and its performance in the study. The reasons for reduction of motility of sperm in the study are not well understood but lack of storing facility could be one of the most important reasons as only one nitrogen can was used for storing frozen sperm and frequent opening of the lid of the can might be the cause of thawing of sperm. the frozen spermatozoa of Labeo rohita, after thawing as effective as the fresh control but the fertilization rate has never exceeded 50% versus the controls, although many more cryopreserved spermatozoa were used.

Knowledge Management: Journal