Investigations on a cryopreservation protocol for long-term storage of psittacine spermatozoa using cockatiel semen as an example

The aim of the present study was the establishment of an effective protocol for cryopreservation of psittacine semen. Therefore, pooled semen samples of 30 cockatiels (Nymphicus hollandicus) were diluted with modified Lake diluent (1:4), partitioned into four equal parts. Three portions were mixed with three cryoprotectants (dimethylacetamide, dimethyl sulfoxide, glycerol) in 4%, 8% and 12% final concentration, respectively, whereas the 4rth part served as control. Altogether, 96 incremental diluted semen samples were obtained for investigation. Each cryoprotective agent (CPA) in each final concentration was evaluated regarding sperm motility immediately after dilution and another four times every 30 min. Sperm viability was evaluated 0 and 120 min after dilution using the fluorescence stain SYBR® Green/propidium iodide. Sperm morphology was evaluated 0 and 120 min after dilution using eosin B stains. Glycerol demonstrated a lethal effect on cockatiel spermatozoa in all concentrations, whereas dimethylacetamide (DMA) in 8% final concentration proved to have the least adverse effect on semen parameters. Comparison of quick and slow freezing methods using DMA 8% revealed significantly higher rates of viable and motile spermatozoa after computer-controlled rate freezing. Two insemination experiments resulted in an egg fertility rate of 92.59% and 67.65% after artificial insemination with freshly collected semen samples, compared to 30.77% and 18.00% egg fertility rates using frozen/thawed semen. Altogether, 12 chicks hatched out of eggs inseminated with cryopreserved semen. To our knowledge, this is the first time for cockatiels to be successfully reproduced after artificial insemination using cryopreserved semen. •A working protocol for the cryopreservation of cockatiel spermatozoa was evaluated.•Using DMA in 8% concentration, combined with SPF, was identified as the most appropriate way to preserve sperm function.•Unsuitable cryoprotective agents and cooling rates have been identified.•Slow programmable freezing (SPF) using a freezing machine resulted in viable spermatozoa after thawing.•Artificial insemination with cryopreserved semen resulted in healthy offspring.