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. 2023 Sep 25;13(19):3014.
doi: 10.3390/ani13193014.

Cryopreservation Cooling Rate Impacts Post-Thaw Sperm Motility and Survival in Litoria booroolongensis

Rebecca J Hobbs  1 , Rose Upton  2 , Natalie E Calatayud  3 , Aimee J Silla  4 , Jonathan Daly  1   5 , Michael S McFadden  1 , Justine K O'Brien  1   5
Affiliations

Cryopreservation Cooling Rate Impacts Post-Thaw Sperm Motility and Survival in Litoria booroolongensis

Rebecca J Hobbs et al. Animals (Basel). .

Abstract

The cryopreservation and storage of gametes (biobanking) can provide a long-term, low-cost option for the preservation of population genetic diversity and is particularly impactful when applied to manage selective breeding within conservation breeding programs (CBPs). This study aimed to develop a sperm cryopreservation protocol for the critically endangered Booroolong frog (Litoria booroolongensis) to capture founder genetics within the recently established (est. 2019) CBP for this species. Hormone-induced sperm release was achieved using established protocols, and spermic urine samples were collected over a 6-h period. Pooled spermic urine samples (n = 3 males) were divided equally between two cryoprotectant (CPA) treatments and diluted by 1:5 (sperm:CPA) with either 15% (v/v) dimethyl sulfoxide + 1% (w/v) sucrose in simplified amphibian Ringer's (SAR; CPAA) or 10% (v/v) dimethylformamide + 10% (w/v) trehalose dihydrate in SAR (CPAB). The samples were cryopreserved in 0.25 mL straws using either a programmable freezer (FrA) or an adapted dry shipper method (FrB). The thawed samples were activated via dilution in water and assessed for viability and motility using both manual assessment and computer-assisted sperm analysis (CASA; 0 h, 0.5 h post-thaw). Upon activation, the survival and recovery of motility (total motility, forward progression and velocity) of cryopreserved sperm suspensions were higher for sperm preserved using FrB than FrA, regardless of CPA composition. This work supports our long-term goal to pioneer the integration of biobanked cryopreserved sperm with population genetic management to maximize restoration program outcomes for Australian amphibian species.

Keywords: ART; CASA; GRB; amphibian; biobanking; breeding program; conservation; reproductive technologies; spermiation.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses or interpretation of the data; in the writing of the manuscript; or in the decision to publish the results.

Figures

Figure A1
Figure A1
Cooling rate curves (FrB) measured for L. booroolongensis sperm biobanking replicates performed at Taronga Conservation Society Australia from 2020 to 2022. Different colors indicate individual “freezing runs”.
Figure 1
Figure 1
Effects of cryoprotectant and cooling rate on sperm total motility (A), membrane integrity (B) and forward-progressive motility (C) at 0 min post-thaw (manual assessment). Sperm motility was assessed in hypo-osmotically activated samples. FrA—programmable, FrB—dry shipper, CPAA—DMSO/SUC, CPAB—DMF/TRE.
Figure 2
Figure 2
Effects of cryoprotectant and cooling rate on sperm velocity parameters at times 0 and 30 min post-thaw (CASA assessment). (A) VCL, curvilinear velocity; (B) VSL, straight-line velocity; (C) VAP, average path velocity. FrA—programmable, FrB—dry shipper, CPAA—DMSO/SUC, CPAB—DMF/TRE.
Figure 3
Figure 3
Average measured cooling curves (mean ± SD) across all experimental replicates. Straws containing CPAA (A,B) show a defined point of thermal fusion between −8 and −14 °C under both cooling conditions (FRA (A), FRB (B) compared to CPAB (C,D).
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References

    1. International Union for Conservation of Nature Species Survival Commission, Amphibian Specialist Group . Amphibian Conservation Action Plan. IUCN; Gland, Switzerland: Cambridge, UK: 2007. p. 64.
    1. International Union for Conservation of Nature Species Survival Commission, Amphibian Specialist Group . The Amphibian Conservation Action Plan (ACAP): A Status Review and Roadmap for Global Amphibian Conservation. Volume Preprint The World Conservation Union (IUCN); Gland, Switzerland: 2022.
    1. Howell L.G., Mawson P.R., Frankham R., Rodger J.C., Upton R.M.O., Witt R.R., Calatayud N.E., Clulow S., Clulow J. Integrating biobanking could produce significant cost benefits and minimise inbreeding for Australian amphibian captive breeding programs. Reprod. Fertil. Dev. 2021;33:573–587. doi: 10.1071/RD21058. - DOI - PubMed
    1. Della Togna G., Howell L.G., Clulow J., Langhorne C.J., Marcec-Greaves R., Calatayud N.E. Evaluating amphibian biobanking and reproduction for captive breeding programs according to the Amphibian Conservation Action Plan objectives. Theriogenology. 2020;150:412–431. doi: 10.1016/j.theriogenology.2020.02.024. - DOI - PubMed
    1. Silla A.J., Calatayud N.E., Trudeau V.L. Amphibian reproductive technologies: Approaches and welfare considerations. Conserv. Physiol. 2021;9:coab011. doi: 10.1093/conphys/coab011. - DOI - PMC - PubMed