Chilled and freeze-dried preservation of saltwater crocodile (Crocodylus porosus) spermatozoa
Patricio D. Palacios, Stephen D. Johnston, Hyemin Kim, David Blyde, John Lever, Andrés GambiniContext
Artificial insemination in farmed crocodiles is limited by poor sperm survival after cryopreservation, requiring alternative preservation strategies.
Aims
This study evaluated the effects of three extenders on saltwater crocodile (Crocodylus porosus) semen during chilled storage for up to 14 days and assessed post freeze-drying (FD) sperm functional integrity.
Methods
Semen was collected from six adult males. Samples were then diluted in a commercial milk-based extender, Hanks’ balanced salt solution (HBSS), or HBSS supplemented with 5% bovine serum albumin (BSA) and stored at 5°C, with sperm assessed at 24 h, 48 h, 72 h, 7 days, and 14 days. Freeze-drying was performed using standard protocols.
Key results
Ejaculate characteristics (mean ± standard error of the mean; s.e.m.) included volume 1.3 ± 0.3 mL, sperm concentration 3.32 ± 0.2 × 109 sperm/mL, total motility 65.0 ± 3.9%, viability 97.8 ± 0.7%, and normal morphology 66.8 ± 6.6%. After 72 h of storage, motility, viability, and normal morphology were maintained across all extenders, with the milk-based extender outperforming the HBSS-based extenders. Cytoplasmic droplets, loose heads, and absent midpieces were the predominant abnormalities observed. Following rehydration, freeze-dried spermatozoa showed viability of 23.0 ± 1.0%, normal morphology of 15.0 ± 3.4%, mitochondrial mass in 55.8 ± 2.7%, and DNA fragmentation of 6.2 ± 0.7%, with no evidence of motility recovered.
Conclusions
Our results suggest that commercial milk-based extenders support chilled storage of crocodile semen for up to 3 days, while representing the first report of spermatozoa FD in a reptile species.
Implications
Chilled semen storage supports semen transport for crocodile artificial insemination or cryopreservation programs, whereas freeze-drying expands future options for reptilian genetic preservation.