The quality of sperm from European cyprinid fish will be assessed to determine whether this can provide a suitable indicator of the effects of pollution on freshwater and manne fish stocks. The effects of pollutants on sperm viability will be examined using Computer Assisted Sperm Analysis (CASA) and electron microscopy and compared with the effects on fertilisation rate. No results are yet available from the project.
Stocks of freshwater and mane fish within the European Union are increasingly threatened by aquatic pollution, but no data are available on the extent by which fertility of individual species has decreased. The aim of the project is to develop a methodology to measure the impact of environmental pollutants on the viability of fish sperm, and to assess the impact of aquatic pollution on fish stocks within the EU.
Recent concern about the effects of pollution has centred on the decrease in human sperm counts and the possibility that this may result from the feminising effects of environmental estrogens (Sharpe and Skakkebak, 1993). The link between estrogenic activity and decreased sperm counts is far from proven and the fact that a wide range of non-estrogenic environmental pollutants can also cause disruption of the reproductive endocrine system that may result in decreased sperm quality is frequently overlooked. Although such disruption may equally well decrease fertility in both sexes, one reason for the emphasis on the male is clearly the much greater ease with which human sperm quality may be assessed compared to egg quality.
The effects of aquatic pollution on the reproductive endocrine system of fish are well documented (Kime, 1995), but there have been very few studies of its effects on gamete quality. While egg quality can only be readily measured by laborious assessment of fertilisation rate and larval survival, sperm quality, as in humans, is amenable to easy measurement. Although there have been a few attempts to quantify the effects of pollutants on sperm motility they have until recently relied on a subjective scoring of either velocity on a 1-5 scale, or duration of movement (Khan and Weis, 1987 a,b; McMaster et al., 1992). Neither method is very well suited to fish sperm which differs in a number of important aspects from that of mammals. Unlike mammals in which sperm is motile on ejaculation, that of fish only becomes motile when it comes into contact with water which changes the surrounding ion coactions. While mammalian sperm remains motile for hours, that of most fish loses movement within minutes after initiation of motility.
Computer Assisted Sperm Analysis (CASA) has been used for some years to assess the motility of nummalian sperm and durmg the last few years has been applied to fish (C!wist et al., 1996; Kime et al., 1996, Rurangwa et al., 1998). Unlike previous methods of assessment it is quantitative, rapid, reproducible, avoids any subjective judgement on the part of the operator and records a large number of parameters of sperm movement. Recent collaborative studies in our laboratories, using the African catfish (Clarias gariepinus) as model species, have shown that CASA has the potential for development as a rapid and reliable quantitative method for assaying the effects of heavy metal pollutants on the motility of fish sperm. A relationship between sperm motility, as assayed by CASA, and fertilisation rate has not been established, but preliminary studies have shown that these are closely correlated. Sperm motility using CASA may therefore provide a simple method both for determining the effects of pollutants on fertility in laboratory studies and for determining whether wild fish populations exposed to polluted waters have become sub-fertile. Our studies have also shown that heavy metals cause a major disruption to sperm morphology as determined by scanning electron microscopy (SEM) which may therefore provide a useful tool to determine pollutant induced damage to the sperm A previous study (Sivarrajah et al., 1978) has also shown that PCBs damage the sperm heads in exposed carp and trout
The overall aim of the project is to provide a simple non-invasive assessment of the effects of aquatic pollution on fish fertility using different parameters of sperm viability that may be used both as a basis of laboratory toxicity tests and for monitoring the reproductive viability of wild populations.
Sperm motility will be measured using Computa Assisted Spsm Analysis of video recordings taken of spsm exposed to pollutants for up to 24 h. Comparisons will be made between the effects recorded by CASA and the rate of in vitro fertilisation of exposed or unexposed sperm and eggs. This requires determination of the minimum sperm:egg ratio for each species chosen, since excess spsm can mask the effects of pollutants (Rurangwa et al., 1998), and will show which species are most at risk in the wild, since those which produce a large excess of sperm will be less vulnsable than those which produce the bare minimum. An artificial model of a spawning fish will be developed so that gametes may be released in similar conditions to that in the wild, and the effects of changing spsm:egg ratios or the effects of pollutants on the fertilising ability of spsm can be monitored. Such a model will permit determination of the species at greatest risk from aquatic pollutants and will have wide applicability to both freshwater and marine fish. Changes induced by pollutants in sperm morphology will be monitored using scanning and transmission electron microscopy. Numbers of live sperm will be determined by histological techniques. To provide a uniform supply of sperm for any particular species, the potential use of cryopreserved sperm will be examined so that samples may be taken from a common stock at any time of the year.
Cyprinid fish (carp, roach, etc) will be used as representatives of the maJor class of teleost fish in European inland waters, but the methodology developed will also be applicable to marine species. For some laboratory-based experiments using natural spawning the zebra-fish (Danio rerio) will also be used. The pollutants chosen will include a sensitive range of heavy metals, organochlorine and organophosphate pesticides, PCBs, PAHs and environmental estrogens.
The early part of the study will use laboratory exposures of sperm, but throughout the project sperm will be taken during the spawning season from wild caught fish in rivers known to contain a variety of pollutants. Collaboration will be initiated with other scientists wiffiin the EU who have laboratory fish exposed to a range of pollutants, or have access to wild populations of freshwater or manne species inhabiting polluted waters, so that the viability of the sperm of such fish may be tested.
In addition to its use in monitoring the long-term effects of pollution on wild fish stocks, the methodology developed will be applied to assessing the quality of sperm from selected broodstock and the stability of cryopreserved stocks.
Christ, S.A. Toth,G.P. Mc Carthy, H.W. Torsella, J.A. and Smith, M.K (1996). Monthly variation in sperm motility in common carp assessed using computer assisted sperm analysis (CASA). J. Fish Biol. 48, 1210-1222.
Khan, A. T. and Weis, J. S. (1987a). Toxic effects of mercuric chloride on sperm and egg viability of two populations of mummichog, Fundulus heteroclitus. Environ. Poll. 48, 263-274.
Khan, A. T. and Weis, J. S. (1987b). Effects of methyl mercury on sperm and egg viability of two populations of killifish (Fundulus heteroclitus). Arch Environ. Contam. Toxicol. 16, 499-506.
Kime, D. E. (1995). The effects of pollution on reproduction in fish. Rev. Fish. Biol. Fisheries 5, 52-96.
Kime, D. E., Ebrahimi, M., Nysten, K., Roelants, I., Rurangwa, E., Moore, H. D. M. and Olivia, F. (1996). Use of computer assisted sperm analysis (CASA) for monitoring the effects of pollution on spe m quality of fish; application to the effects of heavy metals. Aquat. Toxicol. 36, 223-237
McMaster, M. E., Portt, C. B., Munkittick, K. R. and Dixon, D. G. (1992). Milt characteristics, reproductive perforrnance, and larval survival and development of white sucker exposed to krafL rnill effluent. Ecotox. Environ. Saf: 23, 103-117.
Rurangwa, E., Roelants, I., Huyskens, G., Ebrahimi, M., Kime, D. E., and Ollevia, F. (1998). Ille mmnnum effective spermatozoa to egg ratio for artificial insemination and the effects of heavy metal pollutants on sperm motility and fertilization ability.in the African catfish (Clarias gariepinus, Burchell 1822); J. Fish Biol. (In Press).
Sbarpe, R. M. and Skakkebak, N. E. (1993). Are estrogens involved in falling sperm counts and disorders ofthe male reproductive tract. Lancet 341, 1392-1395.
Sivartia}~ K, Franklin, C. S. and Williams, W. P. (1978). Some histopathological effects of Arochlor 1254 on the liver and gonads of rainbow trout, Salmo gairdneri and carp, Cyprinus carpio. J. Fish Biol. 13, 411-414.
Funding SchemeCSC - Cost-sharing contracts