Short-term storage of cane toad (Bufo marinus) gametes.

:The responses of cane toad (Bufo marinus) gametes, used as a model for the development of assisted reproduction techniques for rare and endangered amphibians, to short-term storage at temperatures > 0 degrees C were studied. Whole excised testes were stored at 0 degrees or 4 degrees C for 15 days, and sperm motility was measured at excision and after storage for 2, 5, 7, 10, 12 and 15 days. Spermatozoa showed > 50% motility for 7 days at 0 degrees C and for 5 days at 4 degrees C. At 15 days, only spermatozoa stored at 0 degrees C still showed some motility (3%). Sperm suspensions were prepared at 5 day intervals over 30 days in simplified amphibian ringer (SAR) at dilutions of 1:1, 1:5 and 1:10 (w/v) testes:SAR. Aliquots from each dilution were stored at 0 degrees C in Eppendorf tubes opened at 5 day intervals of storage (aerated) or kept sealed (unaerated) (treatments: aerated or unaerated; 5, 10, 15, 20, 25 and 30 days storage). After 30 days, sperm motility and fertilizing capacity were determined. The optimal protocol for sperm storage up to 10 days, as assessed by the retention of fertilizing capacity, was as a 1:5 testis:SAR (w/v) suspension, whereas the longest absolute retention of both motility and fertilizing capacity was observed in concentrated (1:1 dilution), anaerobic suspensions (up to 25-30 days). Oviductal oocytes placed in SAR at 5, 10, 15, 20 and 25 degrees C immediately after ovulation lost viability when cooled rapidly to 5 degrees C and stored for 2 h. However, oocytes retained viability for up to 8 h at the optimum storage temperature of 15 degrees C. Thus, it is concluded that during short-term storage spermatozoa retain viability for longer than oocytes, and that spermatozoa in suspensions retain viability for longer than spermatozoa stored in situ in excised testes.

Browne RK ，Clulow J ，Mahony M 《REPRODUCTION》

Storage of cane toad (Bufo marinus) sperm for 6 days at 0 degrees C with subsequent cryopreservation.

We investigated the recovery of motility of cane toad (Bufo marinus) sperm after storage for 6 days at 0 degree C (on ice) and after subsequent cryopreservation. Sperm suspensions were prepared from testes macerated in either simplified amphibian Ringer (SAR) or 10% (w/v) sucrose diluents, with 15% or 20% (v/v) glycerol or Me2SO as cryoprotectants, and were stored for 6 days. Alternatively, suspensions were prepared in either SAR or 10% (w/v) sucrose diluent and stored for 6 days, after which some of these suspensions were kept in diluents alone or, alternatively, had 15% or 20% (v/v) glycerol or Me2SO added. All treatments (suspensions) were then cryopreserved. Sperm motility was measured at Day 1 and Day 6 (before and after cryopreservation). A substantial and variable proportion (range 0%-40%) of sperm was immotile in suspensions immediately after preparation from testes macerates. Sperm stored in either SAR or 10% (w/v) sucrose diluent maintained approximately 75% motility for 6 days, but few sperm survived cryopreservation. After storage and cryopreservation, recovery of motility was substantially higher with Me2SO than in glycerol. However, both cryoprotectants exhibited toxicity at high concentrations. Glycerol was more toxic than Me2SO in 10% (w/v) sucrose than in SAR diluent, both before and after cryopreservation. The addition of some cryoprotectants to suspensions before storage gave greater recovery both before and after cryopreservation. After cryopreservation, the highest rate of sperm recovery was in suspensions with 10% (w/v) sucrose and 15% (v/v) Me2SO added prior to storage (mean (+/-SEM) 46 +/- 7% relative to initial; 39 +/- 6% absolute). Sperm were also stored for 6 days at 0 degrees C in suspensions or testes (with suspensions then prepared from testes) and cryopreserved. Sperm maintained higher recovery and membrane integrity both before and after cryopreservation when stored in suspensions rather than in testes.

Browne RK ，Davis J ，Pomering M ，Clulow J ... -  《REPRODUCTION FERTILITY AND DEVELOPMENT》

Optimisation of handling, activation and assessment procedures for Bufo marinus spermatozoa.

In the present study, we investigated handling, activation and assessment procedures for cane toad (Bufo marinus) spermatozoa. Optimisation of these techniques will facilitate the maintenance of sperm viability during cryopreservation and during in vitro fertilisation (IVF) techniques in reproduction technologies for endangered species. Spermatozoa were taken from testicular macerates and assessed using plasma membrane integrity assays (live/dead stains) and quantitative scores of motility parameters. In the assessment of sperm viability using live/dead stains, there were small but significant differences in the percentage of sperm from cryopreserved samples staining positive with propidium iodide, Hoechst H33258 and Trypan blue; these differences were not large and all stains performed acceptably. Spermatozoa were activated by dilution of testicular macerates in water at one of two dilution ratios (1 : 6 or 1 : 20) with or without 0.1-5.0 mM theophylline. Sperm plasma membrane integrity (unstained spermatozoa) was unaffected by either dilution ratio (osmolarity) or theophylline concentration. However, sperm motility was significantly affected by osmolarity and theophylline concentration. The stimulation of sperm motility increased with higher theophylline concentrations and these strongly interacted with lower osmolarities through a higher dilution ratio of sperm macerates with water. Spermatozoa were exposed to increasing centrifugation forces to determine tolerance to physical stresses encountered during washing procedures. Forces between 50 and 800 g were associated with a significant reduction in motility (mean 56 +/- 3% decreasing to 27 +/- 3%), but did not affect staining. In conclusion, centrifugation should be minimised in anuran sperm washing procedures; osmotic shock associated with higher dilution ratios reduces the capacity of anuran sperm to achieve high percentages of motile sperm, leading to a likely trade-off between dilution required for activation and sperm motility to optimise IVF fertilisation rates; and optimal conditions for sperm motility after activation occur at lower dilutions of suspensions with 5.0 mM theophylline. The present study has improved protocols for the handling of anuran sperm during pre- and post-cryopreservation procedures.

Fitzsimmons C ，McLaughlin EA ，Mahony MJ ，Clulow J ... -  《REPRODUCTION FERTILITY AND DEVELOPMENT》

The short-term storage and cryopreservation of spermatozoa from hylid and myobatrachid frogs.

The short-term storage (at 0 degrees C) and cryopreservation of spermatozoa may be useful for providing gametes for fertilisations performed in programmes for the conservation and management of endangered amphibians. The current study was undertaken to examine the applicability of amphibian spermatozoa storage protocols developed with the cane toad (Bufo marinus) to a wider range of amphibian species, with a view to ultimately using these protocols for endangered species. In Australia, at least 29 species of recently extinct or endangered frogs are from the families the Myobatrachidae and the Hylidae. This study investigated the applicability of short-term storage and cryopreservation protocols developed for cane toad (Bufo marinus) spermatozoa to those of hylid and myobatrachid species. Storage of spermatozoa in intact testes or in suspensions for six days at 0 degrees C showed spermatozoa maintained higher motility in suspensions than those in testes, and hylid spermatozoa maintained greater motility than myobatrachid spermatozoa. However, the protocols for optimal storage at 0 degrees C varied with testis size when spermatozoa were stored in whole testes. Spermatozoa from 13 frog species representing both families were cryopreserved using sucrose as diluent with Me(2)SO or glycerol as cryoprotectants. After cryopreservation hylid spermatozoa showed a greater recovery than myobatrachid spermatozoa and Me(2)SO provided higher recovery than glycerol. The freeze-thaw recovery of spermatozoa was independent of testes weight of the species studied. These results show spermatozoa from the Hylidae and Myobatrachidae may be stored both in the short-term (at 0 degrees C) and long-term by cryopreservation using protocols established for B. marinus.

Browne RK ，Clulow J ，Mahony M 《CRYOLETTERS》

Liquid storage of flow cytometrically sorted ram spermatozoa.

This study investigated the optimum short-term storage conditions for ram spermatozoa before and after flow cytometric sorting. Prior to sorting, semen from four rams (n = 3 ejaculates per ram) was diluted in either a Tris-based diluent (TRIS) or AndroHep (AH) and stored at 5, 15 or 21 degrees C for 0, 6 or 24h. Sperm characteristics were assessed during storage and after sorting, freeze-thawing and incubation (6h, 37 degrees C). Functional capacity and migration ability in artificial cervical mucus (sperm migration test (SMT)) of stored, sorted and non-sorted (control) spermatozoa were assessed after freeze-thawing. After sorting, semen from three rams (n = 3 ejaculates per ram) was diluted in four different extenders: ultra-heat-treated (UHT) long life milk, TRIS containing 10% (v/v) egg yolk (TRIS-EY), AH (pH 7.4), or TEST buffer containing 10% (v/v) egg yolk (TYB). Sorted and non-sorted (control) spermatozoa were stored at 15 degrees C for 24h or 5 degrees C for 6 days. Sperm characteristics were evaluated at 0, 6 and 24h for samples stored at 15 degrees C and daily for samples stored at 5 degrees C. The SMT was performed on sorted and non-sorted (control) spermatozoa after 6h and 3 days storage at 15 and 5 degrees C, respectively. Spermatozoa stored in TRIS were sorted more efficiently, had higher motility after sorting, freezing, thawing and incubation and had greater numbers of spermatozoa penetrating into the SMT than spermatozoa stored in AH prior to sorting. Spermatozoa stored in UHT at both temperatures had higher motility, acrosome integrity and traveled greater distances in the SMT than spermatozoa stored in all other diluents. In summary, storage in TRIS at 21 degrees C was optimal for transport of ram spermatozoa to the sorting site, and storage of spermatozoa in UHT diluent (after sorting) preserved sperm viability and migration ability best at both 15 and 5 degrees C.

Hollinshead FK ，O'Brien JK ，Gillan L ，Meyers M ，Maxwell WM ，Evans G ... -  《THERIOGENOLOGY》