NOTE Lowerre-Barbieri and Barbieri: Oocyte separation and preservation for reproduction studies 



169 



oocyte shrinkage (DeMartini Fountain 1981, Schaefer 

 1987, Witthames & Greer Walker 1987), which could 

 mask gaps found naturally in oocyte size-frequency 

 distributions. Gilson's solution also causes continuous 

 shrinkage over time (Witthames & Greer Walker 1987), 

 which could make any comparisons of egg diameter 

 during the spawning season, or between consecutive 

 years, meaningless unless all samples were preserved 

 for the same amount of time. 



Formalin at low concentrations (3-5%) meets the 

 requirements of both an oocyte fixative and preserva- 

 tive (Markle 1984). It prevents microbial activity, with 

 minimal effect on shape, cell contents, and osmolality. 

 As a preservative, it maintains this state, is relatively 

 mild, stable, and long-lasting (Snyder 1983, Markle 

 1984). Although formalin is commonly used to pre- 

 serve ichthyoplankton samples (Snyder 1983), it has 

 not been commonly used for adult fish-reproduction 

 studies. This is due to the tendency for formalin to fix 

 the whole ovary into a hard mass, from which it is 

 difficult to separate individual oocytes ( Schaefer & Or- 

 ange 1956, Bagenal & Braum 1978). 



By physically separating the oocytes before preser- 

 vation in formalin, our method overcomes the problem 

 of oocyte separation while maintaining the advantages 

 of using formalin as a preservative. This method is 

 inexpensive, quick, and much less toxic than Gilson's, 

 providing researchers with undamaged oocytes of all 

 stages, with little effect on appearance or size. This 

 new method has been successfully used on weakfish 

 and two other sciaenids (Atlantic croaker Micro- 

 pogonias undulatus, and black drum Pogonias cromis; 

 unpubl. data), and, given the similarity of teleost ova- 

 ries, should be applicable to a wide range of species. 

 Additionally, because oocytes are preserved in a low 

 concentration of formalin, similar to the preservation 

 of most plankton samples, hydrated oocytes processed 

 in this fashion would be comparable to those collected 

 and preserved during plankton studies. This would 

 make it possible to better link adult fish-reproduction 

 studies with those from egg surveys. 



Acknowledgments 



We would like to thank Sonny Williams for his ex- 

 traordinary help in obtaining hydrated females. Rogerio 

 Teixeira provided helpful insight in the preliminary 

 stages of developing the method. We would also like to 

 thank Mark E. Chittenden Jr., James Colvocoresses, 

 John Graves, John Hunter, Beverly Macewicz, and two 

 anonymous reviewers for reviewing and commenting 

 on the manuscript. Financial support was provided by 

 the College of William and Mary, Virginia Institute of 

 Marine Science and by a Wallop/Breaux Program Grant 



from the U.S. Fish and Wildlife Service through the 

 Virginia Marine Resources Commission for Sport Fish 

 Restoration, Project No. F-88-R3. L.R. Barbieri was 

 partially supported by a scholarship from CNPq, Min- 

 istry of Science and Technology, Brazil (process no. 

 203581/86-OC). 



Citations 



Bagenal, T.B., & E. Braum 



1978 Eggs and early life history. In Bagenal, T. (ed.), 

 Methods for assessment of fish production in fresh- 

 water, p. 165-201. IBP (Int. Biol. Programme) 

 Handb. 3. 



Brown-Peterson, N„ P. Thomas, & C.R. Arnold 



1988 Reproductive biology of the spotted seatrout, 

 Cynoscion nebulosus, in south Texas. Fish. Bull., U.S. 

 86:373-388. 



Cailliet, G.M., M.S. Love, & A.W. Ebeling 



1986 Fishes. Wadsworth Publ. Co., Belmont CA, 194 p. 

 Clark, F.N. 



1934 Maturity of the California sardine (Sardina 

 caerulea), determined by ova diameter measure- 

 ments. Calif. Fish Bull. 42:1-49. 

 DeMartini, E.E. 



1990 Annual variations in fecundity, egg size and con- 

 dition of the plainfin midshipman iPorichthys nota- 

 tus). Copeia 1990:850-855. 

 DeMartini, E.E., & R.K. Fountain 



1981 Ovarian cycling frequency and batch fecundity in 

 the queenfish, Seriphus politus: Attributes represen- 

 tative of serial spawning fishes. Fish. Bull., U.S. 

 79:547-560. 

 Draper, N.R., & H. Smith 



1981 Applied regression analysis, 2d ed. John Wiley, 

 NY, 709 p. 

 Fleming, I. A., & S. Ng 



1987 Evaluation of techniques for fixing, preserving, 

 and measuring salmon eggs. Can. J. Fish. Aquat. 

 Sci. 44:1957-1962. 



Hiemstra, W.H. 



1962 A correlation table as an aid for identifying pe- 

 lagic fish eggs in plankton samples. J. Cons. Perm. 

 Int. Explor. Mer 27:100-108. 

 Hislop, J.R.G., & M.A. Bell 



1987 Observations on the size, dry weight and energy 

 content of the eggs of some demersal fish species from 

 British marine waters. J. Fish Biol. 31:1-20. 

 Hunter, J.R. 



1985 Preservation of northern anchovy in formalde- 

 hyde solution. In Lasker, R. (ed.), An egg production 

 method for estimating spawning biomass of pelagic 

 fish: Application to the northern anchovy, Engraulis 

 mordax, p. 63-64. NOAA Tech. Rep. NMFS 36. 

 Hunter, J.R., & S.R. Goldberg 



1980 Spawning incidence and batch fecundity in north- 

 ern anchovy, Engraulis mordax. Fish. Bull., U.S. 

 77:641-652. 



