Sperm cryopreservation of African catfish, Clarias gariepinus: Cryoprotectants, freezing rates and sperm : egg dilution ratio

Methods for cryopreserving spermatozoa and optimizing sperm:egg dilution ra tio in African catfish Clarias gariepinus were developed. Five percent to 2 5% DMSO and methanol were tested as cryoprotectants, by diluting semen in G inzburg fish ringer and freezing in 1-milliliter cryovials in a programmabl e freezer. To avoid an excess of spermatozoa per egg, post-thaw semen was d iluted 1:20, 1:200 or 1:2000 before fertilization. Highest hatching rates w ere obtained by spermatozoa frozen in 10% methanol and post-thaw diluted to 1:200. Then, slow freezing rates (-2, -5 or -10 degreesC/min) to various e ndpoint temperatures (range -25 to -70 degreesC) before fast freezing in li quid nitrogen (LN2) were evaluated. Hatching rates equal to control (P>0.05 ) were obtained by spermatozoa frozen at -5 degreesC/min to -45 to -50 degr eesC and at -10 degreesC/min to -55 degreesC. In 3-step freezing programs, at -5 degreesC/min, the effect of holding spermatozoa for 0, 2 or 5 min at -30, -35 or -40 degreesC before fast freezing in LN2 was analyzed. Hatching rates equal to control (P>0.05) were produced by spermatozoa frozen to, an d held at, -35 degreesC for 5 min and at -40 degreesC for 2 or 5 min. Final ly, frozen spermatozoa (10% methanol, -5 degreesC/min, 5-min hold at -40 de greesC, LN2, post-thaw diluted to 1:200) were tested in on-farm fertilizati on conditions. Again, no difference (P>0.05) in hatching rate was observed between frozen and fresh spermatozoa. Cryopreservation offers utility as a routine method of sperm storage and management for catfish. (C) 2000 by Els evier Science Inc.