Effect of sperm concentration, medium osmolality and oocyte storage on artificial fertilisation success in a myobatrachid frog (Limnodynastes tasmaniensis)
D. L. Edwards A C , M. J. Mahony B and J. Clulow BA School of Animal Biology, University of Western Australia, 35 Stirling Hwy, Crawley, WA 6009, Australia.
B School of Life and Environmental Sciences, University of Newcastle, University Drive, Callaghan, NSW 2308, Australia.
C To whom correspondence should be addressed. email: dan@cyllene.uwa.edu.au
Reproduction, Fertility and Development 16(3) 347-354 https://doi.org/10.1071/RD02079
Submitted: 18 September 2002 Accepted: 23 February 2004 Published: 25 May 2004
Abstract
The present study optimised artificial fertilisation and oocyte storage conditions in Limnodynastes tasmaniensis (Myobatrachidae). Data on general reproductive biology, the effect of sperm motility and concentration, medium osmolality and oocyte storage on artificial fertilisation success are presented. Egg number was most strongly correlated with bodyweight (r = 0.819). Sperm yield was correlated with testes weight (r = 0.827), which was strongly correlated with snout–vent length (r = 0.772). Optimal artificial fertilisation occurred in 0–7 mOsm kg–1 amphibian Ringer, similar to ranid, bufonid and hylid species. High fertilisation rates were achieved using spermatozoa with little forwards progressive motility at comparatively low concentrations (3 × 104 sperm cells mL–1) and with no relationship between percentage sperm motility and fertilisation success (correlation of fertilisation rate with sperm motility after activation: r = –0.145). Oocytes stored in 5 mOsm kg–1 solutions showed no significant decline in fertilisability after 2 h, showing that swelling of the jelly surrounding the eggs does not prevent sperm from fusing with the oocyte in this species. Fertilisability of oocytes was extended to > 4 h in medium to high osmolality solutions (124–271 mOsm kg–1). These data allow for the future use of L. tasmaniensis in developing assisted reproductive technology protocols for foam-nesting myobatrachid species, many of which are now threatened with extinction in the wild.
Acknowledgments
The authors would like to thank J. D. Roberts and A. Hettyey for helping to improve earlier versions of this paper.
Alford, R. A. , and Richards, S. J. (1999). Global amphibian declines: a problem in applied ecology. Annu. Rev. Ecol. Syst. 30, 133–165.
| Crossref | GoogleScholarGoogle Scholar |
Barberi, F. D. , and Raisman, J. S. (1969). Non-gametic factors involved in the fertilization of Bufo arenarum oocytes. Embryologica 10, 363–372.
Bawa, K. S. , Menon, S. , and Gorman, L. R. (1997). Cloning and conservation of biological diversity: paradox, panacea, or Pandora’s box? Conserv. Biol. 11, 829–830.
| Crossref | GoogleScholarGoogle Scholar |
Benirschke, K. (1984). The frozen zoo concept. Zoo Biol. 3, 325–328.
Browne, R. K. , Clulow, J. , Mahony, M. , and Clark, A. (1998). Successful recovery of motility and fertility of cryopreserved cane toad (Bufo marinus) sperm. Cryobiology 37, 339–345.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Browne, R. K. , Mahony, M. J. , and Clulow, J. (2002). The short-term storage and cryopreservation of spermatozoa from hylid and myobatrachid frogs. Cryo. Letters 23, 129–136.
| PubMed |
Buhler, M. I. , and Zelarayan, L. (1997). Influence of ovulation conditions upon the metabolism and cleavage capacity in Bufo arenarum oocytes. J. Exp. Zool. 278, 101–105.
| Crossref | GoogleScholarGoogle Scholar |
Byrne, P. G. , Roberts, J. D. , and Simmons, L. W. (2002). Sperm competition for increased testes mass in Australian frogs. J. Evol. Biol. 15, 347–355.
| Crossref | GoogleScholarGoogle Scholar |
Caputo, M. , Infante, V. , Talevi, R. , Vaccaro, M. C. , Carotenuto, R. , and Campanella, C. (2001). Following passage through the oviduct, the coelomic envelope of Discoglossus pictus (Amphibia) acquires fertilizability upon reorganization, conversion of gp 42 to gp 40, extensive glycosylation, and formation of a specific layer. Mol. Reprod. Dev. 58, 318–329.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Clark, A. K. (1994). Spermatozoa motility in Hylidae and Myobatrachidae frogs. Honours Thesis. (University of Newcastle: Newcastle, Australia.)
Clulow, J., Mahony, M., Browne, R. K., Pomering, M. and Clark, A. (1999). Applications of assisted reproductive technologies (ART) to endangered anuran amphibians. In
Cogger, H. G. (1992).
Cross, N. L. , and Elinson, R. P. (1980). A fast block to polyspermy in frogs mediated by changes in the membrane potential. Dev. Biol. 75, 187–198.
| PubMed |
del Pino, E. (1973). Interactions between gametes and environment in the toad Xenopus laevis (Daudin) and their relationship to fertilization. J. Exp. Zool. 185, 121–132.
Elinson, R. P. (1975). Site of sperm entry and a cortical contraction associated with egg activation in the frog Rana pipiens. Dev. Biol. 47, 257–268.
| PubMed |
Elinson, R. P. (1977). Fertilization of immature frog eggs: cleavage and development following subsequent activation. J. Embryol. Exp. Morphol. 37, 187–201.
| PubMed |
Elinson, R. P. (1986). Fertilization in amphibians: the ancestry of the block to polyspermy. Int. Rev. Cytol. 101, 59–94.
| PubMed |
Emmens, C. W. (1947). The motility and viability of rabbit spermatozoa at different hydrogen-ion concentrations. J. Physiol. 106, 471–481.
Gosner, K. L. (1960). A simplified table for staging anuran embryos and larvae with notes on identification. Herpetologica 16, 477–480.
Hollinger, T. G. , and Corton, G. L. (1980). Artificial fertilization of gametes from the South African clawed frog, Xenopus laevis. Gamete Res. 3, 45–57.
Holt, W. V. , Bennett, P. M. , and Volobouev, V. (1996). Genetic resource banks in wildlife conservation. J. Zool. 238, 531–544.
Horton, P. (1982). Precocious reproduction in the Australian frog Limnodynastes tasmaniensis. Herpetologica 38, 486–489.
Katagiri, C. (1961). On the fertilizability of the frog egg I. J. Fac. Sci. Hokkaido Univ., Ser. VI. Zool. 14, 607–613.
Katagiri, C. (1962). On the fertilizability of the frog egg II. Change of the jelly envelopes in water. Jpn. J. Zool. 13, 365–373.
Kuramota, M. (1978). Correlations of quantitative parameters of fecundity in amphibians. Evolution 32, 287–296.
Lofts, B. (1974). Reproduction. In
Lee, M. S. Y. , and Jamieson, B. G. M. (1992). The ultrastructure of the spermatozoa of three species of Myobatrachid frogs (Anura, Amphibia) with phylogenetic considerations. Acta Zoologica 73, 213–222.
Mahony, M., Clulow, J., Browne, R. K. and Pomering, M. (1999). Declines and disappearances of frogs: risk assessment and contingency strategies. In
Morisawa, M. and Morisawa, S. (1990). Acquisition and initiation of sperm motility. In
Moriya, M. (1976). Required salt concentration for successful fertilization of Xenopus laevis. J. Fac. Sci. Hokkaido Univ., Ser. VI. Zool. 20, 272–276.
Olson, J. H. , and Chandler, D. E. (1999). Xenopus laevis egg jelly contains small proteins that are essential to fertilization. Dev. Biol. 210, 401–410.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Omata, S. (1993). Relative roles of jelly layers in successful fertilization of Bufo japonicus. J. Exp. Zool. 265, 329–335.
Pettus, D. , and Angleton, G. M. (1967). Comparative reproductive biology of montane and piedmont chorus frogs. Evolution 21, 500–507.
Ralls, K. , and Ballou, J. D. (1986). Preface to the proceedings of the workshop on genetic management of captive populations. Zoo Biol. 5, 81–86.
Rugh, R. (1937). A quantitative analysis of the pituitary-ovulation relation in the frog (Rana pipiens). Physiol. Zool. 10, 84–100.
Rugh, R. (1962).
Salthe, S. N. and Mecham, J. S. (1974). Reproductive and courtship patterns. In
Schäuble, C. S. , Moritz, C. , and Slade, R. W. (2000). A molecular phylogeny for the frog genus Limnodynastes (Anura: Myobatrachidae). Mol. Phylogenet. Evol. 16, 379–391.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Seymour, R. S. , and Roberts, J. D. (1991). Embryonic respiration and oxygen distribution in foamy and nonfoamy egg masses of the frog Limnodynastes tasmaniensis. Physiol. Zool. 64, 1322–1340.
Wolf, D. P. , and Hendrick, J. L. (1971). A molecular approach to fertilisation. II. Viability and artificial fertilisation of Xenopus laevis gametes. Dev. Biol. 25, 360–376.
| PubMed |
