Chilled and freeze-dried preservation of saltwater crocodile (Crocodylus porosus) spermatozoa

Abstract

CONTEXT: 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.