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Establishment of Cryo-milt Bank for Carps and Catfishes

Dr. Md. Khalilur Rahman
Chief Scientific Officer
Bangladesh Fisheries Research Institute

Abstract/ Executive Summary:

Healthy male of Pangasius hypophthalmus and Labeo rohita were selected during peak breeding season. Male breeders were hypophysed with cPGE at the rate of 2 mg per kg of body weight to get milt easily. Milt was collected after 6 hrs of hypophysation. Mill was collected from the male by stripping into several ice cold sterilized tubes. Separate tubes were used for P. hypophthalmus and L. rohita, respectively. Sterilised tubes containing milt were stored in ice box for 2, 4 and 8 hours. Motility of Spermatozoa was assessed by a microscope. Motility rate of Pangasius hypophthalmus ranged between 5 and 50% and fertilisation rate varied between 1 and 40%, respectively. Hatching rates were very poor that ranged between 0 and 20%. On the other hand, experiments were also conducted with short-term preserved milt and fresh eggs of Labeo rohita. Motility rate of Labeo rohita ranged from 5 to 40% while fertilisation rate varied between 1 and 20%, respectively. Poor hatching rates were recorded with the short-term preserved milt of L. rohita that ranged between 0 and 15%

Keywords: Cryo-preservation, Milt, Pangasius hypophthalmus, Labeo rohita

Location:

Start Date:

End Date:

Program Area: Variety and Species

Commodity: Fish

Objectives:

The main objective of this project is to establish Cryo-Milt Bank for Carps and Catfishes. Other objectives are as follows:

Developing appropriate collection techniques and transport systems of fish milt
Cryo-Milt Bank for Labeo rohita and Pangasius hypophthalmus (sutchi)
Estimation of breeding success of L. rohita and P. sutchi using cryo-milt

Methodology:

Mature and healthy male brood fishes were selected to collect milt. Milt sample were expelled from the male fish by gentle abdominal pressure and collected into a clean and dry tube of 5 ml capacity. The milt samples were primarily placed in sealed ice box for 2-3 minutes then it was stored in a normal freezer for 20 minutes at 0 to 4⁰C and then milt was stored in a deep freezer at -15⁰C. Before storage, cryo-diluent was prepared. Dimethyl-sulphoxide (DMSO) was used as cryo-protectant and phosphate buffered saline was used as extender. Cryo-diluents solution was prepared by mixing 75% phosphate buffered saline, 10% DMSO and 15% skimmed milk. This composition was homogenously mixture by vortex mixture. Milt was stored in 5 ml cryo-tubes. Cryo-tubes were leveled as A, B and C for storing 4, 8 and 12 hours, respectively. The milt was mixed with the solution at the ratio 1:1(v/v); and stored in 5 ml leveled cry-tubes. This cry tubes ware gently shaken to allow milt to properly mix with solution, and then carry those cryo-tubes by ice box. Leveled cry-tubes ware placed in the refrigerator for 15 min to reduce their temperature to 4°C before fast freezing in the freezer. Then this tubes ware storage in the -15°C deep freezer. In the thawing process, this cryo tubes ware first transferred into refrigerator for 20 min to allow the milt to thaw from -15°C to 4°C inside the refrigerator. There after, they ware transferred at room temperature and allowed to stand 5 min before mixing with female aggs. The sperm motility rates of the freshly collected semen sample were evaluated prior to cryopreservation. Sperm motility of fresh milt was recorded as 95%.Samples of milt were removed from the deep freezer. The 5 ml tube was hold in a water bath at a temperature of 37-40°C for around 7-10 seconds. As a general rule, samples are thawed when air bubbles within the straw can move freely within the liquid. Thawed milt was added to the eggs of Pangasius hypophthalmus and thoroughly mixed. After approximately 5 minutes, water was added to water-harden the eggs.

Source of Information: Annual Report. 2017-19. 232 p.

Article Link (Online Journal):

Funding Source:

1. Government

Budget: 500,000

Total Budget (Tk.) : NaN

Achievement/Findings:

Results of the experiments with short-term preserved milt and fresh eggs of P. hypophthalmus and L. rohita were not satisfactory for commercial operation of fish hatcheries. However, it is a preliminary result and needs modification of short-term preservation method. Therefore, more experiments are to be conducted for getting scientifically explainable results on short-term preservation of fish milt in Bangladesh condition.

Knowledge Management: Report/Proceedings