Cryopreservation of the sperm of the Japanese bitterling

Abstract

Sperm of the Japanese bitterling Tanakia limbata that had been cryopreserved with 5 or 10% methanol plus 95 or 90% foetal bovine serum (FBS) showed higher percentage and longer duration of motility than those that had been cryopreserved with 90% FBS and 10% DMSO, glycerol, N,N-dimethylacetamide or N,N-dimethylformamide. Foetal bovine serum, used as extender, had some cryoprotective effects when spermatozoa were cooled either with 10% methanol or without methanol. Spermatozoa, cooled to 40 C and then immersed in liquid nitrogen, had greater post-thaw motility than those cooled to 20, 60, or 80 C. The post-thaw percentage of motile spermatozoa increased significantly (P<0·001) with decreases in the freezing rate from 60 to 5 C min 1 . These results indicate that 10% methanol plus 90% foetal bovine serum is a suitable diluent for cryopreservation of bitterling spermatozoa and that samples should be cooled to 40 C at a low freezing rate for effective storage. 2001 The Fisheries Society of the British Isles

Sperm motility and cryopreservation of spermatozoa in freshwater gobies

“…Several studies have shown FBS to be an excellent cryopreservation diluent (Aoki et al , 1997; Ohta et al , 2001; Tsutaka et al , 2006). In the present study, no conspicuous differences in post‐thaw motility were found between ASP and FBS when 10% MeOH was used as a cryoprotectant.…”

“…Optimum cooling rates for spermatozoa vary among species; for example, from −5 to −15° C min −1 in the oily bitterling Tanakia limbata (Temminck & Schlegel) (Ohta et al , 2001), −40° C min −1 in the Colorado pikeminnow Ptychocheilus lucius Girard (Tiersch et al , 2004) and −42·5° C min –1 in the ayu Plecoglossus altivelis altivelis (Temminck & Schlegel) (Tsutaka et al , 2006). Tiersch (2001) recommended a cooling rate of −40° C min –1 for sperm cryopreservation in a broad range of species.…”

“…Spermatozoa were considered motile when the sperm head showed forward movement in consecutive video frames from 5 to 10 s. Per cent motility was measured by tracing the location of heads of >50 randomly chosen spermatozoa on a TV monitor (Ohta et al , 2001). Measurements at each dilution were repeated twice using two tubes, and the average result used in data analysis.…”

“…The monitoring tube was placed near the sample tubes in each experiment. Cooling rates were calculated based on the time required for the temperature to change from 0 to −40° C (Ohta et al , 2001).…”

“…Cryopreservation techniques can offer an economical and specific tool for conserving the genetic resources of endangered species. Although methods for sperm cryopreservation have been reported in many fish species, principally in large, commercial species such as salmonids and marine teleosts (Lahnsteiner, 2000; Suquet et al , 2000; Chao & Liao, 2001), few studies have addressed the subject in tiny endangered fishes (Aoki et al , 1997; Ohta et al , 2001).…”