Influence of Xenon on Survival of Sperm of Common Frog Rana temporaria during Slow Freezing.

We studied the effect of xenon on the survival rate of the spermatozoa of the common frog Rana temporaria during slow freezing with saturation of the suspension with xenon at a pressure of up to 1.2 bar. The cryoprotective properties of xenon were analyzed in comparison with nitrogen. No specific cryoprotective effect of xenon was revealed. Viability of spermatozoa pretreated with xenon at atmospheric pressure (0 bar) or under excess pressure of 0.6 bar and frozen in a cryoprotective medium with dimethylformamide, sucrose, and BSA did not differ significantly. The use of overpressure of xenon of 1.0 or 1.2 bar in the pretreatment and freezing process significantly impaired viability of the biomaterial.

Sperm motility of externally fertilizing fish and amphibians.

We review the phylogeny, sperm competition, morphology, physiology, and fertilization environments of the sperm of externally fertilizing fish and amphibians. Increased sperm competition in both fish and anurans generally increases sperm numbers, sperm length, and energy reserves. The difference between the internal osmolarity and iconicity of sperm cells and those of the aquatic medium control the activation, longevity, and velocity of sperm motility. Hypo-osmolarity of the aquatic medium activates the motility of freshwater fish and amphibian sperm and hyperosmolarity activates the motility of marine fish sperm. The average longevity of the motility of marine fish sperm (~550 seconds) was significantly (P < 0.05) greater than that of freshwater fish sperm (~150 seconds), with the longevities of both marine and freshwater fish being significantly (P < 0.05) lower than that of anuran sperm (~4100 seconds). The average velocity of anuran sperm (25 µm/s) was significantly (P < 0.05) lower than that of marine fish (140 µm/s) or freshwater fish (135 µm/s) sperm. The longevity of the sperm of giant salamanders (Cryptobranchoidea) of approximately 600 seconds was greater than that of freshwater fish sperm but much lower than anuran sperm. Our research and information from the literature showed that higher osmolarities promote greater longevity in anuran sperm, and some freshwater fish sperm, and that anuran and cryptobranchid sperm maintained membrane integrity long after the cessation of motility, demonstrating a preferential sharing of energy reserves toward the maintenance of membrane integrity. The maintenance of the membrane integrity of anuran sperm in fresh water for up to 6 hours showed an extremely high osmotic tolerance relative to fish sperm. The very high longevity and osmotic tolerance of anuran sperm and high longevity of cryptobranchid sperm, relative to those of freshwater fish, may reflect the complex fertilization history of amphibian sperm in general and anurans reversion from internal to external fertilization. Our findings provide a greater understanding of the reproductive biology of externally fertilizing fish and amphibians, and a biological foundation for the further development of reproduction technologies for their sustainable management.

Hormonal induction of spermatozoa from amphibians with Rana temporaria and Bufo bufo as anuran models.

The use of hormonally induced spermatozoa expressed in urine (HISu) is a valuable component of reproduction technologies for amphibians. Five protocols for sampling HISu from the European common frog (Rana temporaria) were compared: (1) pituitary extracts, (2) 0.12 µg g⁻¹ luteinising hormone-releasing hormone analogue (LHRHa), (3) 1.20 µg g⁻¹ LHRHa, (4) 11.7 IU g⁻¹ human chorionic gonadotrophin (hCG) and (5) 23.4 IU g⁻¹ hCG (g⁻¹ = per gram bodyweight). From 1 to 24h after administration we assessed the number and concentration of spermatozoa in spermic urine and in holding water, and in urine the percentage of motile spermatozoa and their progressive motility. The protocol using 1.20 µg g⁻¹ LHRHa gave the highest total sperm numbers (650 × 106) and the highest percentage (40%) of samples with sperm concentrations above 200 × 106 mL⁻¹. The percentage motility and progressive motility was similar from all protocols. Considerable amounts of spermatozoa were expressed by R. temporaria into their holding water. We tested hormonal priming and spermiation in the common toad (Bufo bufo) using 0.13 µg g⁻¹ LHRHa administered 24h before a final spermiating dose of 12.8 IU g⁻¹ hCG. No spermatozoa were expressed in holding water. Priming resulted in 35% more spermatozoa than without; however, there were no differences in sperm concentrations. Primed B. bufo produced spermatozoa with significantly higher percentage motility, but not progressive motility, membrane integrity, or abnormal spermatozoa than unprimed males.

Cryopreservation of hormonally induced sperm for the conservation of threatened amphibians with Rana temporaria as a model research species.

The survival of hundreds of threatened amphibian species is increasingly dependent on conservation breeding programs (CBPs). However, there is an ongoing loss of genetic variation in CBPs for most amphibians, reptiles, birds, and mammals. Low genetic variation results in the failure of CBPs to provide genetically competent individuals for release in supplementation or rehabitation programs. In contrast, in the aquaculture of fish the perpetuation of genetic variation and the production of large numbers of genetically competent individuals for release is accomplished through the cryopreservation of sperm. Successful protocols for the cryopreservation of amphibian sperm from excised testes, and the use of motile frozen then thawed sperm for fertilisation, have been adapted from those used with fish. However, there have been no protocols published for the cryopreservation of amphibian hormonally induced sperm (HIS) that have achieved fertility. We investigated protocols for the cryopreservation of amphibian HIS with the European common frog (Rana temporaria) as a model research species. We induced spermiation in R. temporaria through the intraperitoneal administration of 50 µg LHRHa and sampled HIS through expression in spermic urine. Highly motile HIS at a concentration of 200 × 10(6)/mL was then mixed 1:1 with cryodiluents to form cryosuspensions. Initial studies showed that; 1) concentrations of ∼15 × 10(6)/mL of HIS achieve maximum fertilisation, 2) TRIS buffer in cryodiluents did not improve the recovery of sperm after cryopreservation, and 3) high concentrations of DMSO (dimethylsulphoxide) cryoprotectant reduce egg and larval survival. We then compared four optimised cryopreservation protocols for HIS with the final concentrations of cryodiluents in cryosuspensions of; 1) DMSO, (½ Ringer Solution (RS), 10% sucrose, 12% DMSO); 2) DMSO/egg yolk, (½ RS, 10% sucrose, 12% DMSO, 10% egg yolk), 3) DMFA, (½ RS, 10% sucrose, 12% dimethylformamide (DMFA)), and 4) MIS/glycerol, (Motility Inhibiting Saline (MIS), 5% glycerol, 2.5% sucrose, 5% egg yolk). Cryosuspensions were frozen in LN(2) vapour, stored in LN(2), thawed in 40° C water bath, and activated by slow equilibration with 1:3 dilutions of cryosuspensions with 20 mM/L NaCl. Protocol efficacies were assessed through the post-thaw percentage of; 1) sperm motility, 2) sperm membrane integrity, 3) fertilisation, 4) fertilised eggs hatching, and 5) larval survival from fertilised eggs to 7 d. The DMFA cryodiluent proved superior to the DMSO based cryodiluents in recovery of sperm motility and fertility after cryopreservation. MIS/glycerol cryodiluent provided low sperm viability and no fertility. Considering the ease of obtaining HIS from many Rana species, the success of our protocols offer the potential for the perpetuation of the genetic variation of the 42 threatened Rana species and the 193 threatened Ranid species in total.

[Fluorescent analysis of cryopreserved totipotent cells of amphibian embryos]. / Fluorestsentnyi analiz kriokonservirovannykh totipotentnykh kletok zarodyshei amfibii.

The cryotolerance of totipotent cells from dissociated embryos of amphibian (grass frog Rana temporaria and grey toad Bufo bufo) was studied. Cell integrity and preservation of the cell barrier function were evaluated by fluorescent analysis. It was shown that the best cryopreservation of the cells was achieved by using the cryoprotective agent 10% dimethyl sulfoxide and 10% saccharose. These cells were successfully used for the homotransplantation of nuclei into enucleated eggs. The development of reconstructed eggs to the blastula stage was noted.