Md. Nahiduzzaman

Sperm cryopreservation of the critically endangered olive barb (Sarpunti) Puntius sarana (Hamilton, 1822)

The present study focused on development of a sperm cryopreservation protocol for the critically endangered olive barb Puntiussarana (Hamilton, 1822) collected from two stocks within Bangladesh and reared in the Fisheries Field Laboratory, Bangladesh Agricultural University (BAU). The sperm were collected in Alsevers solution prepared at 296mOsmol kg(-1). Sperm were activated with distilled water (24mOsmol kg(-1)) to characterize motility. Maximum motility (90%) was observed within 15s after activation, and sperm remained motile for 35s. Sperm activation was evaluated in different osmolalities and motility was completely inhibited when osmolality of the extender was ≥287mOsmol kg(-1). To evaluate cryoprotectant toxicity, sperm were equilibrated with 5%, 10% and 15% each of dimethyl sulfoxide (DMSO) and methanol. Sperm motility was noticeably reduced within 10min, when sperm were equilibrated with 15% DMSO, indicating acute toxicity to spermatozoa and therefore this concentration was excluded in further trials. Sperm were cryopreserved using DMSO at concentrations of 5% and 10% and methanol at 5%, 10% and 15%. The one-step freezing protocol (from 5°C to -80°C at 10°C/min) was carried out in a computer-controlled freezer (FREEZE CONTROL® CL-3300; Australia) and 0.25-ml straws containing spermatozoa were stored in liquid nitrogen for 7-15days at -196°C. The highest motility in thawed sperm 61±8% (mean±SD) was obtained with 10% DMSO. The fertilization and hatching rates were 70% and 37% for cryopreserved sperm, and 72% and 62% for fresh sperm. The protocol reported here can be useful for hatchery-scale production of olive barb. The use of cryopreserved sperm can facilitate hatchery operations, and can provide for long-term conservation of genetic resources to contribute in the recovery of critically endangered fish such as the olive barb.

Sperm cryopreservation of the Indian major carp, Labeo calbasu: Effects of cryoprotectants, cooling rates and thawing rates on egg fertilization

A sperm cryopreservation protocol for the Indian major carp, Labeo calbasu, was developed for long-term preservation and artificial fertilization. Milt collected from mature male fish were placed in Alsevers solution (296mOsmolkg(-1)) to immobilize the sperm. Cryoprotectant toxicity was evaluated by motility assessment with dimethyl sulfoxide (DMSO) and methanol at 5, 10 and 15% concentrations. DMSO was more toxic at higher concentrations than methanol, and consequently 15% DMSO was excluded from further study. A one-step cooling protocol (from 5 to 80°C) with two cooling rates (5 and 10°C/min) was carried out in a computer-controlled freezer (FREEZE CONTROL(®) CL-3300; Australia). Based on post-thaw motility, the 10°C/min cooling rate with either 10% DMSO or 10% methanol yielded significantly higher (P=0.011) post-thaw motility than the other rate and cryoprotectant concentrations. Sperm thawed at 40°C for 15s and fresh sperm were used to fertilize freshly collected L. calbasu eggs and significant differences were observed (P=0.001) in percent fertilization between cryopreserved and fresh sperm as well as among different sperm-to-egg ratios (P=0.001). The highest fertilization and hatching rates were observed for thawed sperm at a sperm-to-egg ratio of 4.1×10(5):1. The cryopreservation protocol developed can facilitate hatchery operations and long-term conservation of genetic resources of L. calbasu.

Bottleneck in the endangered kalibaus, Labeo calbasu (cyprinidae: cypriniformes) populations in Bangladesh revealed by microsatellite DNA marker analysis

Microsatellite DNA marker analysis was carried out to assess the population genetic structure of an endangered carp, Labeo calbasu, collected from three different stocks; the Jamuna River, the Halda River and a Hatchery. Four heterologous microsatellite loci (Lr12, Lr14b, Lr21 and Lr24) identified from rohu (Labeo rohita) were analyzed to test the genetic variability of the target kalibaus stocks. The maximum number of alleles observed in loci Lr12, Lr14b, Lr21 and Lr24 were 10, 7, 8 and 6, respectively. The loci were found to be polymorphic (<P95) in all the populations. The average number of allele was highest in the Jamuna population (6.75) followed by that of the Halda (5.50) and the Hatchery population (4.25). The observed average heterozygosity (Ho) value was almost similar in all three populations. Except locus Lr12 in the Halda population, significant deviations from the Hardy-Weinberg Equilibrium were detected in all cases due to excess heterozygosity. The population differentiation values (FST) between all the population pairs were significant. The highest genetic distance value (D = 0.295) was measured between the Halda and the Hatchery populations. A recent bottleneck was observed in the Halda and the Hatchery population.

Threatened fishes of the world: Puntius sarana (Hamilton 1822) (Cyprinidae)

Common name: Olive barb (English). Conservation status: Critically endangered in Bangladesh (IUCN 2000), vulnerable category in India (Mijkherjee et al. 2002). Identification: DIII+8, AIII+5, V9, P15. The largest species in this genus (Fig. 1), a moderately compressed body with two pairs of barbels, lateral line scales 32–34. Maximum length 42 cm, and 1.4 kg (Rahman 2005). Distribution: Widely distributed in Bangladesh, India, Nepal, Bhutan, Myanmar and Thailand (Talwar and Jhingran 1991). Abundance: Very low abundance in a few rivers and floodplains of Bangladesh. Habitat and ecology: Inhabits mostly freshwater and occasionally brackish water. It feeds on aquatic insects, algae and shrimp (Riede 2004). Reproduction: Mature fish (at least 1 year old) breed during April-September (Sinha 1973; Khanam et al. 2008), in running, shallow waters with submerged vegetation. Threats: Poor survival of egg and larvae (Chondar 1999), over-exploitation and habitat degradation (Chakraborty et al. 2003). Conservation action: Some permanent fish sanctuaries have been established in the deeper portions of some rivers and floodplains (Jamuna and the Brahmaputra basins). Several initiatives have been undertaken to promote the species among the farmers after developing aquaculture techniques (Chakraborty et al. 2005). The development of ‘cryopreservation protocol’ for olive barb sperm is under way in Bangladesh. Conservation recommendations: Sustainable fisheries management plan along with mass awareness and conservation based re-stocking program need to be implemented. Remarks: The olive barb is a popular food fish having high market demand in Bangladesh and in other South Asian countries (Jena et al. 2007). Its introduction into carp polyculture system would not only help in diversification of aquaculture Environ Biol Fish (2010) 87:197–198 DOI 10.1007/s10641-010-9579-z