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Effect of Storage Time on Cryopreseved Sperm of an Endangered Fish, Labeo Calbasu

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

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

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

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

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

Abstract/ Executive Summary:

Sperm collected from mature male kalibaus, Labeo calbasu was examined up to ten months after cryopreservation with the view to evaluate the effect of storage time on sperm viability. Sperm was collected in 0.9% NaCl or Alsever solution and the volume (µl g^-1), concentration (×10¹⁰ml^-1) and pH were determined. Sperm samples were diluted (sperm : cryoprotectant : extender - 1 : 1 : 8) and equilibrated for 8-10 minutes. The software Cryogenesis^TMV5 at one-step cooling (5 ^oC to – 80^oC at 10 ^oC/min) was used to freeze the sample. Motility of thawed sperm lasted for 45 s after activation with distilled water (24 mOsmol kg^-1). The post-thaw sperm motility (%) in two combinations were found to be 82 ± 8 and 75 ± 5 at the end of 1^st fortnight and 70 ± 10 and 67 ± 6 after 20^th fortnight. One of the major cause of reduced motility of sperm over time was multipurpose use of same liquid nitrogen dewar where the sperms were preserved.

Keywords: Labeo calbasu, Cryopreservation, Sperm viability, Cryoprotectant

Location: Kustia,Chittagong and Bogra

Start Date: 00-00-0000

End Date: 00-00-0000

Program Area: Animal Health and Management

Commodity: Fish

Objectives:

To determine the effect of liquid nitrogen storage on the viability of Labeo calbasu sperm.

Methodology:

Broods were collected from the three rivers - the Padma (from Kustia district), the Halda (from Chittagong district) and the Jamuna (from Bogra district). Collected broods were reared in the brood rearing ponds of the Fisheries Field Laboratory Complex, Bangladesh Agricultural University (BAU), Mymensingh. Prior to milt collection, mature males were selected and immediately carried to the Mini Hatchery of Faculty of Fisheries. After conditioning, males were treated with carp pituitary supernatant (2 mg kg^?1 body weight) for the induction of maturation and spermiation. Fish were caught after 6 hours of PG injection and a gentle pressure was applied on the abdomen for collecting the sperm. Samples were collected in modified Alsevre’s solution (0.7 g NaCl plus 0.8 g sodium citrate dissolved in 100 ml water; osmolality, 296 mosmol kg^-1). The collected samples were kept in ice (4^oC) until use. After collection, initial quality of sperm samples were checked under a light microscope (Novex K-range, Holland) at × 400 magnifications. During motility (%) estimation, 1 ml milt sample was activated with 19 ml distilled water (24 mOsmol kg^-1) on glass slide. Sperm that had active forward movement was considered as motile. Sperm sample showed >85% motility after activation was used for cryopreservation. Sperm concentration was determined using haemacytometer (Germany) and expressed as number of cells×10¹⁰ml^-1. pH of sperm was determined with a pH indicator strips (pH: 0–14; Merck, Germany). Two different extenders were used in the study to dilute the sperm sample prior to cryopreservation. Dimethyl sulfoxide (DMSO) was used as cryoprotectant and combined with both of the extenders. Collected samples were diluted using the following ratio (sperm : cryoprotectant: extender - 1 : 1 : 8) and equilibrated for 8-10 minutes. The pre-labeled 0.25 ml plastic straws (Tiefenbach, Germany) were filled with 230 µl diluted sperm and sealed with hand sealer. Computer controlled freezer (Cryologic, Australia, FREEZE CONTROL® CL-3300) with a software (CryoGenesis^TMV5) at one-step freezing (5^oC to – 80^oC at a rate of 10^oC) was used to freeze the sample after plunging the straws into the cryochamber. After freezing the straws were retrieved from the cryochamber and kept in liquid nitrogen (-196ºC). After storage in liquid nitrogen (-196ºC) the motility of the cryopreserved sperm was studied fortnightly (three replications each) up to ten months. During motility (%) observation, samples were thawed at 40ºC for 7 seconds in water bath and taken under microscope (Novex K-range, Holland) at × 400 magnifications. Post-thaw percent motility values were arc-sine square root transferred and analyzed with two factors ANOVA where fortnights and combinations were fixed factors. Means were separated by Duncan’s Multiple Range Test and considered significantly different at P < 0.05 (11.5 windows version).

Source of Information: Bangladesh J. Agriculturist. 3(1): 2009 ISSN 1812-4631

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

The percentage of motility of spermatozoa may have decreased due to use of same liquid nitrogen chamber for several purposes and retrieving of canisters for observation at different time. Kumar (1989) found 20-80% and 5-60% motility by using egg-yolk citrate and urea-egg yolk extenders during the storage period of 10 days. Multipurpose use of the same liquid nitrogen dewar and frequent retrieving of canisters reduce the sperm viability. Therefore, Labeo calbasu sperm requires proper management during liquid nitrogen storage after cryopreservation to be viable for an indefinite time as well as to maintain future conservation options.

Knowledge Management: Journal