22 Comparison of conventional and controlled-rate freezing methods to cryopreserve white spotted bamboo shark (Chiloscyllium plagiosum) sperm: implications for elasmobranch biobanking efforts

At least 25% of elasmobranch species are threatened with extinction. Biobanking of sperm and application of assisted reproductive techniques can aid in the genetic management of populations. To date there have been limited sperm cryopreservation studies conducted in elasmobranch species. The present study investigated the use of a conventional freezing method in liquid nitrogen (LN₂) vapour, a controlled-rate freezer, and two cryoprotecting agents, glycerol or dimethyl sulfoxide (DMSO), to cryopreserve white spotted bamboo shark (Chiloscyllium plagiosum) sperm. Ejaculates (n = 9) were manually expressed and held at 5°C before analysis. A 10-µL aliquot of raw ejaculate was extended 1:100 (v:v) with artificial sea water to activate motility. Plasma membrane integrity (PMI) was assessed by staining diluted cells with 200 nM Sybr-14 and 24 µM propidium iodide and counting 200 cells. Spermatozoa were assessed as percent plasma membrane intact (green) or disrupted (red or partially red). Raw ejaculates were extended 1:1 (v:v) with Hanks’ elasmobranch Ringer solution containing 20% egg yolk (ER-EY) at 5°C. Split samples were extended 1:1 (v:v) over 10 min with a ER-EY solution containing 20% glycerol or dimethyl sulfoxide, loaded into 0.5-mL straws and cryopreserved conventionally (∼45°C min⁻¹) or using a controlled-rate freezer (2°C min⁻¹) before plunging into LN₂ (−196°C) and storing for at least 24 h. Straws were thawed in a 20°C water bath and diluted 1:1 (v:v) with ER-EY over 10 min. Descriptive statistics were used to summarise motility and PMI parameters pre-freeze and post-thaw. A two-way ANOVA was used to compare the effect of cryopreservation method and CPA on PMI post-thaw using Genstat 19th edition (VSN International). Raw motility was assessed as 71.7 ± 6.7% and PMI as 72.3 ± 6.4%. A decrease in PMI was observed with the addition of glycerol (42.3 ± 6.3%) and DMSO (46.4 ± 7.9%) at 5°C. Cryopreservation using conventional freezing did not yield any motile sperm post-thaw; controlled-rate freezing resulted in 5.0 ± 1.0% motility in samples cryopreserved with DMSO but 0% with glycerol. Controlled-rate freezing resulted in higher (P < 0.001) PMI (24.9 ± 3.3%) than conventionally cryopreservation (3.0 ± 2.1%). There were no differences (P > 0.05) in PMI between cryoprotecting agents post-thaw for conventionally frozen samples (glycerol 4.4 ± 3.1%; DMSO 0.25 ± 0.25%) or controlled-rate frozen samples (glycerol 22.0 ± 2.9%; DMSO 27.9 ± 8.6%). Results indicate that cryopreservation has a profound effect on motility, similar to findings in other elasmobranch species. Increased sperm survival using a controlled-rate freezer emphasises the importance of cooling rate when cryopreserving elasmobranch sperm. Further optimisation of cryoprotecting agents and media that maintains motility post-thaw is required to effectively biobank sperm and genetically manage elasmobranch populations.