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Uteshev V. K., Gakhova E. N., Kramarova L. I., Shishova N. V., Kaurova S. A. New Approaches to Collecting Reproductive Material from Amphibians for its Use in Artificial Fertilization. Current Studies in Herpetology, 2019, vol. 19, iss. , pp. 46-55. DOI:

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New Approaches to Collecting Reproductive Material from Amphibians for its Use in Artificial Fertilization


This paper describes new methods for obtaining viable ovulated oocytes and testicular sperm from the carcasses of females and males of the common frog Rana temporaria, stored at +4ºС for 1–7 days. In addition, a new approach to delayed collection (1 to 30 days) of ovulated oocytes from live female frogs of the same species is given. Part of the frog testicular spermatozoa is shown to retain motility (21.0±1.5%) and fertilizing ability (13.2±1.9%) even after 6 days of storage at +4°C in the carcasses of males. Ovulated oocytes stored in female frog carcasses at 4°C for eight days retained the ability for fertilization (39.2±4.2%) and subsequent development until hatching (16.0±6.2%). Our results also indicate the possi-bility of delayed (up to 30 days) in vivo obtaining oocytes capable to fertilization (46.4±3.0%) and further development until hatching (49.2±7.7%). The results of this paper are a further step in the development of modern reproductive technologies.


Dabagyan N. V., Sleptsova L. A. Common frog Rana temporaria L. In: E. A. Astaurov and T. A. Detlaf, eds. Objects for Developmental Biology. Moscow, Nauka Publ., 1975, pp. 422–462 (in Russian).
Kaurova S. A., Chekurova N. R., Melnikova E. V., Uteshev V. K., Gakhova E. N. Cryopreservation of frog Rana temporaria sperm without loss of fertilizing capacity. In: Genetic Resource Conservation: Proc. of XIV Working Meeting. Pushchino, Institut biofiziki kletki RАN Publ., 1996, pp. 106–108 (in Russian).
Kaurova S. A., Uteshev V. K., Gakhova E. N. Cryopreservation of testicular sperm of common toad Bufo bufo. Biophysics of living cells. Genetic Resource Conservation, 2008, vol. 9, pp. 62 (in Russian).
Ananjeva N. B., Uteshev V. K., Orlov N. L., Ryabov S. A., Gakhova E. N., Kaurova S. A., Kramarova L. I., Shishova N. V., Browne R. K. Comparison of the modern reproductive technologies for amphibians and reptiles. Russian J. of Herpetology, 2017, vol. 24, no. 4, pp. 1–16.
Blaustein A. R., Kiesecker J. M. Complexity in conservation: lessons from the global decline of amphibian populations. Ecology Letters, 2002, vol. 5, pp. 597–608.
Browne R. K., Clulow J., Mahony M., Clark A. Successful recovery of motility and fertility of cryopreserved cane toad (Bufo marinus) sperm. Cryobiology, 1998, vol. 37, iss. 4, pp. 339–345. 
Browne R. K., Clulow J., Mahony M. Short-term storage of cane toad (Bufo marinus) gametes. Reproduction, 2001, vol. 121, no. 1, pp. 167–173.
Browne R. K., Clulow J., Mahony M. The short-term storage and cryopreservation of spermatozoa from hylid and myobatrachid frogs. CryoLetters, 2002, vol. 23, no. 2, pp. 129–136.
Clulow J., Clulow S. Cryopreservation and other assisted reproductive technologies for the conservation of threatened amphibians and reptiles: bringing the ARTs up to Speed. Reproduction, Fertility and Development, 2016, vol. 28, no. 8, pp. 1116–1132.
Kouba A., Vance C., Calatayud N., Rowlison T., Langhorne C., Willard S. Assisted Reproduction Technologies (ART) for Amphibians. In: V. A. Poole, S. Grow, eds. Amphibian Husbandry Resource Guide. Maryland, Silver Spring, 2012, pp. 60–118.
Kouba A. J., Vance C. K. Applied reproductive technologies and genetic resource banking for amphibian conservation. Reproduction, Fertility and Development, 2013, vol. 21, no. 6, pp. 719–737.
Langhorne C. J., Calatayud N. E., Kouba A. J., Feugang J. M., Vance C. K., Willard S. T. Cryoconservation: Successful sperm cryopreservation and developmental outcomes using endangered North American amphibians. Cryobiology, 2013, vol. 67, no. 3, pp. 405–409.
Mansour N., Lahnsteiner F., Patzner R. A. Optimization of the cryopreservation of African clawed frog (Xenopus laevis) sperm. Theriogenology, 2009, vol. 72, iss. 9, pp. 1221–1228.
Mansour N., Lahnsteiner F., Patzner R. A. Motility and cryopreservation of spermatozoa of European common frog, Rana temporaria. Theriogenology, 2010, vol. 74, iss. 5, pp. 724–732. 
Mugnano J. A., Costanzo J. P., Beesley S. G., Lee R. E. Evaluation of glycerol and dimethyl sulfoxide for the cryopreservation of spermatozoa from the wood frog (Rana sylvatica). CryoLetters, 1998, vol. 19, pp. 249–254.
Rugh R. Induced ovulation and artificial fertilization in the frog. The Biological Bulletin, 1934, vol. 66, pp. 22–29. 
Rugh R. Experimental Embryology, Techniques and Procedures. Minneapolis, Burgess Publishing Company, 1962. 526 p. 
Shishova N. R., Uteshev V. K., Sirota N. P., Kuznetsova E. A., Kaurova S. A., Browne R. K., Gakhova E. N. The quality and fertility of sperm collected from European common frog (Rana temporaria) carcasses refrigerated for up to 7 days. ZooBiology, 2013, vol. 32, iss. 4, pp. 400–406.
Stuart S. N., Chanson J. S., Cox N. A., Young B. E., Rodrigues A. S., Fischman D. L., Waller R. W. Status and trends of amphibian declines and extinctions world-wide. Science, 2004, vol. 306, pp. 1783–1786.
Uteshev V. K., Shishova N. V., Kaurova S. A., Manokhin A. A., Gakhova E. N. Collection and cryopre-servation of hormonally induced sperm of pool frog (Pelophylax lessonae). Russian J. of Herpetology, 2013, vol. 20, no. 2, pp. 105–109.
Uteshev V. K., Gakhova E. N., Kramarova L. I., Shishova N. V., Kaurova S. A., Browne R. Refrigerated storage of European common frog Rana temporaria oocytes. Cryobiology, 2018, vol. 83, pp. 56–59.

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