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Cryopreservation of Sperm of Labeo Rohita (hamilton, 1822) and Its Use in Fertilization and Hatching of Eggs

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

A. K. M. A. Rahman
Department of Fisheries Biology and Genetics, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

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

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

M. K. J. Bhuiyan
Department of Agricultural Statistics, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

Abstract/ Executive Summary:

Cryopreservation is one of the widely accepted and useful methods for preservation of gamete especially the spermatozoa. Experiments were conducted to develop the protocols for cryopreservation of sperm of Labeo rohita and to assess the effect of cryopreserved sperm on fertilization and hatching. Six extenders Alsever’s solution, egg-yolk citrate, urea egg-yolk, Kurokura-2, M^a and M^b and five cryoprotectants ethanol, methanol, DMSO, DMA and glycerol were employed. Diluents were prepared by mixing the cryoprotectants at 10% concentration of the extenders (% v/v). Milt and diluents were mixed at a ratio of 1:9 for Alsever’s solution and Kurokura-2; and 1:4 for urea egg-yolk, egg-yolk citrate, M^a and M^b. Egg-yolk citrate with 10% DMSO showed best performance producing 85 ± 1.6% post-thaw motility followed by 83±2% and 81±2.9% with Alsever’s solution and urea egg-yolk respectively. Other extenders did not produce satisfactory results. Milt was diluted at six ratios (1:2, 1:4, 1:7, 1:9, 1:15 and 1:20) and 1:4 dilution showed the highest post-thaw motility for egg-yolk citrate and urea egg-yolk and 1:9 for Alsever’s solution. Six cryoprotectant concentrations (5, 7, 10, 15, 20% and 30%) were investigated and 10% concentration produced the highest post-thaw motility. In breeding trials, sperm preserved with egg-yolk citrate plus DMSO as well as Alsever’s solution plus DMSO fertilized eggs and produced hatchlings. The fertilization and hatching rates were 57 ± 7% and 46.5 ± 3.5% for egg-yolk citrate, and 33.5 ± 3.5% and 27±3% for Alsever’s solution respectively. Fresh sperm yielded 76 ± 4% fertilization and 70.5 ± 5.5% hatching. The protocol developed through this study can be applied for long-term conservation of genetic materials of L. rohita and the cryopreserved sperm can be used in artificial breeding for generating new individuals.

Keywords: Labeo rohita, Spermatozoa, Cryopreservation, Fertilization, Hatching

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

Start Date:

End Date:

Program Area: Variety and Species

Commodity: Carp fish

Objectives:

To determine the suitable extenders and cryoprotectants, milt dilution ratio, cryoprotectant concentration and fertilizing capability of cryopreserved sperm.

Methodology:

Representative numbers of mature brood fish of L. rohita were collected from different sources (Halda river origin and from different government hatcheries) and stocked in the ponds located in the northern side of the Fisheries Faculty Building, Bangladesh Agricultural University, Mymensingh. The weight of the individuals ranged from 2 to 4 kg. Fish were reared with supplementary feed consisting of mustard oil cake, rice bran, wheat bran, and fish meal at the rate of 30%, 30%, 30% and 10% respectively. Required amount of Megavit-aqua (vitamin-mineral-amino acid premix) was supplemented with the feed to enhance the gonadal development of fish. The feed was administered twice daily at the rate of 4-5% of the body weight. Organic (cow dung) and inorganic (urea and phosphate) fertilizers were also applied at a rate of 2 kg/dec and 150g/dec respectively, at 15 days interval to increase the natural food production of the pond. Regular adding of underground water, liming (250g/dec/month), checking of fish health and water quality monitoring were also done during the experimental period. Male and female brood fish were collected from rearing ponds and kept in cisterns in the hatchery for acclimatization through proper showering and aeration. To determine the suitable combination of extenders and cryoprotectants, six extenders Alsever’s solution, egg-yolk citrate, urea egg-yolk, Kurokura-2, M^a and M^bsolutions and five cryoprotectants methanol, ethanol, DMSO, DMA, and glycerol were used. Data of the experiments I, II and III were presented as percentage of motile cells. The effects of different extenders and cryoprotectants on equilibration and post-thaw motility of sperm, dilution ratios and cryoprotectant concentrations were analyzed using two factor randomized block design through computer software package (MSTAT). Means were separated by Least Significant Difference (LSD) Test and DMRT at 1% and 5% level of significance. The effects of cryopreserved sperm on fertilization and hatching were analyzed using paired t-Test.

Source of Information: Progress. Agric. 22(1 & 2): 123 – 137, 2011 ISSN 1017-8139

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

The preliminary attempts those were taken to develop protocol for cryopreservation of sperm of L. rohita seemed successful. Fertilization and hatching of eggs using cryopreserved sperm has been possible but further improvement is necessary. Although the cryopreservation protocol developed in the experiment is useful for conserving biodiversity of L. rohita, few points such as improvement of fertilization techniques, suitable egg sperm ratio, and feasibility of cryogenic gene banking need to be investigated.

Knowledge Management: Journal