Development of a cryopreservation method for marine and freshwater teleost spermatozoa

M. Ohta, T. Unuma (Reproduction Div.), and K. Kawamura (Genetics Div.)


  A common method for cryopreservation of sperm of both a marine (Japanese eel) and a freshwater spawning teleost (Japanese bitterling) was developed. We investigated and compared the effects of various factors, such as cryoprotectants extenders, equilibration time, activating solutions, temperature before immersion in liquid nitrogen (LN), and cooling rates on post-thaw motility of the spermatozoa of both species spermatozoa were compared.


[Objective]
  Techniques of sperm cryopreservation have been established for various benefits such as (1) Transport of gametes, (2) Systematic mating for maintenance of genetic diversity in the cultured stock, (3) Storage of spermatozoa of uniform quality for evaluating egg quality, (4) Utilization of individual genes with high and economical value. In addition, we are trying to develop methods for conserving the genetic resourced of threatened and endangered teleosts using combinations of sperm cryopreservation with androgenesis, in which embryonic chromosomes are derived only from spermatozoa.
[Results]
  Sperm cryopreserved with the diluent constituted of 10% methanol plus 90% fetal bovine serum (FBS) showed a higher percentage and longer duration of motility than those that had been cryopreserved with 90% FBS and 10% of other representative cryoprotective agents (Figs. 1 and 2). Spermatozoa, cooled to -40℃ and then immediately immersed in LN, had a greater post-thaw percentage of motile spermatozoa than those cooled -20, -60, or -80℃. The post-thaw percentage of motile spermatozoa increased significantly with decreases in the freezing rate from -70 to -5℃/min (Figs. 3 and 4). This trend was similar in the spermatozoa of both freshwater teleost (Japanese bitterling) and sea-spawner teleost (Japanese eel) which are taxonomically greatly different. Thus the method for cryopreservation of spermatozoa obtained in our experiments (10% methanol plus 90% FBS as a cryopreservation diluent and samples should be cooled to -40℃ at a low freezing rate) seems to be applicable for many teleost species.
[Publications]
1) Ohta et al., (2001). Cryopreservation of the sperm of the Japanese bitterling. J. Fish Biol., 58, 670-681.
2) Ohta et al., (2001). Control by the environmental concentration of ions of the potential for motility in Japanese eel spermatozoa. Aquaculture, 198, 339-351.
3) Gwo et al., (1999). Cryopreservation of sperm from the endangered Formosan landlocked salmon. Theriogenology, 51, 569-582. 

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