Trehalose and Dimethyl Sulfoxide As Cryoprotective Solution Inclusions in a Static Liquid Nitrogen Vapour Vitrification Method for African Catfish (Clarias gariepinus Burchell, 1822) Sperm

Captive breeding of Clarias gariepinus (Burchell, 1822) entails stripping of mature females, while males must be killed or testes partially excised to obtain spermatozoa. Cryopreservation could significantly improve the reproductive potential of male catfish. In this study, Ginzburg fish Ringer (GFR) was the common extender in all protocols with or without 0.3 M trehalose. Dimethyl sulfoxide (DMSO) and methanol were tested to determine the preferred equilibration period for the complex before cryofreezing. The cryopreservation method and sperm fertilisation capacity were assessed in terms of hatchability rate. A feasible semen rationing protocol for maximised fertilisation was also evaluated. Semen equilibration with cryoprotectants was allowed for 30 minutes at 4 °C. Liquid nitrogen (LN2) vapour-induced freezing rates ranged from -2.7 to -5 °C.min⁻¹, and cryovials/straws were immersed in LN2 when -41 °C was reached after 12 min. Straws showed significantly improved cryopreservation efficacy over cryovials in terms of fertilisation. Optimal fertilisation occurred with trehalose and DMSO-treated cryostored sperm, comparable to fresh semen. Only 0.05 mL diluted semen was required to fertilise 35g eggs, compared with a similar volume efficacy for undiluted sperm. Trehalose enhanced egg fertilisation for both diluted fresh sperm and DMSO-treated thawed sperm. All methods produced healthy larvae to 21 days post-hatch. The validated method can yield 60 straws (0.5 mL) from 3 mL semen, fertilising approximately 2.1 kg eggs (~162 × 10⁴ eggs). The LN2 vapor vitrification method was shown to be both as effective as and more cost efficient than controlled rate freezing equipment.