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.

[Prospects for Application of Gases and Gas Hydrates to Cryopreservation].

In the present review, we tried to evaluate the known properties of gas hydrates and gases participating in the formation of gas hydrates from the point of view of the mechanisms of cryoinjury and cryoprotection, to consider the papers on freezing biological materials in the presence of inert gases, and to analyze the perspectives for the development of this direction. For the purpose, we searched for the information on the physical properties of gases and gas hydrates, compared processes occured during the formation of gas hydrates and water ice, analyzed the influence of the formation and growth of gas hydrates on the structure of biological objects. We prepared a short review on the biological effects of xenon, krypton, argon, carbon dioxide, hydrogen sulfide, and carbon monoxide especially on hypothermal conditions and probable application of these properties in cryopreservation technologies. The description of the existing experiments on cryopreservation of biological objects with the use of gases was analyzed. On the basis of the information we found, the most perspective directions of work in the field of cryopreservation of biological objects with the use of gases were outlined. An attempt was made to forecast the potential problems in this field.

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.

Viability of postmortal epididymal mouse spermatozoa during long-term hypothermic storage and cryopreservation.

The study examined the effect of long-term hypothermic (4°C) storage of mouse carcasses on motility, cell membrane damage, in vitro survival and capacitation of epididymal spermatozoa before and after cryopreservation. It was shown that the number of spermatozoa with rectilinear forward motion decreased with increasing storage time. There were no significant changes in the total sperm motility and integrity of their plasmalemma. Pronounced effects of hypothermia and long-term storage of the mouse carcasses on cryocapacitation of spermatozoa during cryoconservation were demonstrated.

[Slowing down the rate of irreversible age-related atrophy of the thymus gland by atopic autotransplantation of its tissue, subjected to long-term cryoconservation].

An experimental procedure has been developed enabling to slow down the rate of irreversible atrophy of the thymus gland. The atopic autotransplantation of its tissue subjected to prolonged cryoconservation enables one to inhibit the aging of the organism with respect to several biochemical and immunological indicators.

Localization of green fluorescent protein in mouse preimplantation embryos.

The localization of enhanced green fluorescent protein (EGFP) was studied in preimplantation embryos obtained from reciprocal mating of hemizygous C57Bl/6-Tgn (ACTbEGFP)1Osb/J mice with C57Bl/6 mice. Specific fluorescence of EGFP was observed in all oocytes and embryos obtained from transgenic females during all preimplantation stages and in embryos inheriting the EGFP gene from transgenic males starting from the 8 blastomere stage during the compactization period. EGFP mRNA or EGFP synthesized during oogenesis can be retained in embryos during the entire preimplantation period, while expression of EGFP gene transferred from the father coincides with the onset of compactization. The possibility of using these embryos in experimental mammalian embryology is discussed.

[Atopically transplanted thymus tissues are capable of distantly changing the metabolic status of the body].

It has been found that the atopic transplantation of the thymus tissue from young animals in different variants is capable of significantly decreasing the rate of irreversible age-related atrophy of the thymus, which retards the age-dependent degradation of the T-cell unit of the immune system of the organism.

[The conservation of the genetic resources of deer (Cervidae) by the cryopreservation of their germ cells]. / Sokhranenie geneticheskikh resursov olen'ikh (Cervidae) putem kriokonservatsii ikh polovykh kletok.

Necessity of organization of a cryobank for the sperm of some taxa has been substantiated on the basis of the published data and authors' results concerning the state of genetic resources of the family Cervidae in Russia. A task has also been set to elucidate some factors that may affect the success of cryoconservation, such as the age of male donors; season; duration of sperm storage before and after freezing, including long-term storage; and the possibility of obtaining of sperm from perished males. Results are provided on the influence of these factors of the quality of spermatozoa obtained on three species of the Cervidae.