title: Sperm cryopreservation of African catfish, Clarias gariepinus: cryoprotectants, freezing rates and sperm:egg dilution ratio.
abstract: Methods for cryopreserving spermatozoa and optimizing sperm:egg dilution ratio in African catfish Clarias gariepinus were developed. Five percent to 25% DMSO and methanol were tested as cryoprotectants, by diluting semen in Ginzburg fish ringer and freezing in 1-milliliter cryovials in a programmable freezer. To avoid an excess of spermatozoa per egg, post-thaw semen was diluted 1:20, 1:200 or 1:2,000 before fertilization. Highest hatching rates were obtained by spermatozoa frozen in 10% methanol and post-thaw diluted to 1:200. Then, slow freezing rates (-2, -5 or -10 degrees C/min) to various endpoint temperatures (range -25 to -70 degrees C) before fast freezing in liquid nitrogen (LN2) were evaluated. Hatching rates equal to control (P > 0.05) were obtained by spermatozoa frozen at -5 degrees C/min to -45 to -50 degrees C and at -10 degrees C/min to -55 degrees C. In 3-step freezing programs, at -5 degrees C/min, the effect of holding spermatozoa for 0, 2 or 5 min at -30, -35 or -40 degrees C before fast freezing in LN2 was analyzed. Hatching rates equal to control (P > 0.05) were produced by spermatozoa frozen to, and held at, -35 degrees C for 5 min and at -40 degrees C for 2 or 5 min. Finally, frozen spermatozoa (10% methanol, -5 degrees C/min, 5-min hold at -40 degrees C, LN2, post-thaw diluted to 1:200) were tested in on-farm fertilization 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.
authors.affiliation: Fish Culture and Fisheries Group, Wageningen University, The Netherlands. Ana.Viveiros@alg.venv.wau.nl
authors.forename: A T
authors.initials: AT
authors.lastname: Viveiros
authors.forename: N
authors.initials: N
authors.lastname: So
authors.forename: J
authors.initials: J
authors.lastname: Komen
mesh_terms: D000818:Animals; D002397:Catfishes; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents; D005260:Female; D005615:Freezing; D008297:Male; D000432:Methanol; D009584:Nitrogen; D010063:Ovum / Q000502:physiology*; D012662:Semen Preservation; D012996:Solutions; D013076:Sperm Count; D013094:Spermatozoa / Q000502:physiology*; D013997:Time Factors
chemical_list: D003451:Cryoprotective Agents; D012996:Solutions; D009584:Nitrogen; D000432:Methanol