546 



Fishery Bulletin 105(4) 



such a substantial difference showing an opposite trend 

 is somewhat surprising and is probably largely due to 

 the difference in freezing methods. Although freezing 

 ovarian material in water may improve the quality of 

 the preserved oocytes, it is probably responsible for the 

 large and variable changes that we observed. Because 

 ovarian material is placed in hypotonic distilled water, 

 it will absorb the water, thus increasing the size and 

 weight of the oocytes. If it takes a long time for ovar- 

 ian material in water to become isotonic, the material 

 may not be osmotically stable by the time it is frozen 

 and even by the time the oocytes are measured, which 

 could cause the observed changes and variation in size 

 and weight. In future research ovarian material should 

 be frozen in an isotonic solution to prevent oocytes from 

 swelling. 



The initial reason ovary samples were preserved in 

 a variety of treatments was to determine if oocytes 

 preserved by these methods could be used in digital 

 image analysis. The only studies we are aware of that 

 employ digital image analysis to count and measure fish 

 oocytes preserve them in either formalin (Thorsen and 

 Kjesbu, 2001; Yoneda and Wright, 2004) or modified 

 Gilson's solution (Friedland et al., 2005). All treatments 

 in our study preserved ovarian material of cod, had- 

 dock, and plaice well enough to permit easy identifca- 

 tion and measurements of oocytes. Clumping was a fac- 

 tor for many samples in all but the Gilson's treatment, 

 but most clumps could be broken up well by shaking 

 the vial for 30-60 seconds. Very tight clumps could 

 be broken apart by rapidly drawing in and expelling 

 oocytes and solution for 30-120 seconds with a 10-mL 

 glass pipette with an =1 mm diameter and a plastic, 

 thumb-wheel pipette pump. A glass pipette worked bet- 

 ter than a plastic transfer pipette, probably because its 

 rigidity allows more suction to be created as fluid is 

 quickly drawn into it. With simple, albeit occasionally 

 time-consuming (up to five minutes) mechanical separa- 

 tion, all clumps could be sufficiently broken up so that 

 the oocytes could be measured and counted. 



Considering all comparisons for all three species, cod 

 and haddock oocytes and ovarian material are affected 

 similarly by a given type of preservative, whereas effect 

 on plaice tends to differ with different preservatives. 

 This is not unexpected because cod and haddock are 

 in the same family, Gadidae, whereas the narrowest 

 taxonomic group common to cod and plaice is the sub- 

 division Euteleostei. One would expect tissues of closely 

 related species would have similar chemical properties, 

 and should thus be affected similarly by preservatives. 

 Although it could be inferred from results among stud- 

 ies that differences in preservation exist among fish 

 species, specific differences have not been reported be- 

 fore the present study. 



A difference in the effect of preservation between cod 

 samples from GB and those from GOM is also reported. 

 This difference is unexpected but is supported by the 

 results of comparisons of oocyte size in the Gilson's, 

 freezing, and split-formalin treatments, and by the com- 

 parison of ovarian material weight in the lobe-formalin 



treatment. Samples from GB were collected in Febru- 

 ary, whereas samples from the GOM were collected in 

 May, which may somehow contribute to this difference. 

 Still, it is unclear why time of year or region of sample 

 collection would affect preservation and we suggest this 

 as an area worthy of future research. 



We successfully evaluated and quantified the ef- 

 fects of several preservatives on the ovarian material 

 of several fish species, but perhaps more importantly 

 we demonstrated how preservation can add variation 

 to seemingly simple measurements like ovary weight 

 and oocyte size, and can have different effects between 

 species. Thus we stress the importance of consistent 

 experimental methods and suggest that in studies of 

 preserved ovarian material it should not be assumed 

 that the effects of different preservatives and preserva- 

 tion treatments are consistent. 



Acknowledgments 



We thank R. Brown, J. Ford, R. Sherman, B. Hoffman, 

 M. Russo and T. Ligenza for providing us with access to 

 ovary samples. We also thank K. Friedland, E. Garcia- 

 Vazquez, O. S. Kjesbu, two anonymous reviewers, A. 

 Moles, and E. Calvert for their suggestions and com- 

 ments. Funding for this study was provided through the 

 Cooperative Marine Education and Research Program 

 (CMER) at the University of Massachusetts, Amherst 

 and a Hatch grant. Writing was completed while F. 

 Juanes was a Center Fellow at the National Center 

 for Ecological Analysis and Synthesis, funded by NSF 

 (Grant #DEB-0553768), the University of California, 

 Santa Barbara, and the State of California. 



Literature cited 



Black, R., and S. I. Dodson. 



2003. Ethanol: a better preservation technique for 

 Daphnia. Limnol. Oceanogr. Methods. 1:45-50. 

 Boreman, J. L., B. S. Nakashima, J. A. Wilson, and R. L. 

 Kendall. 



1997. Northwest Atlantic groundfish: perspectives on 

 a fishery collapse, 242 p. Am. Fish. See, Bethesda, 

 MD. 

 Fleming, I. A., and S. Ng. 



1987. Evaluation of techniques for fixing, preserving 

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

 44:1957-1962. 

 Friedland, K. D., D. Ama-Abasi, M. Manning, L. Clarke, 

 G. Kligys, and R. C. Chambers. 



2005. Automated egg counting and sizing from scanned 

 images: Rapid sample processing and large data volumes 

 for fecundity estimates. J. Sea Res. 54:307-316. 

 Joseph, J. 



1963. Fecundity of yellowfin tuna {Thunnus albacares) 

 and skipjack (Katsuwonus pelamis) from the eastern 

 Pacific Ocean. Inter-Am. Trop. Tuna Comm. Bull. 

 7:255-292. 

 Kjesbu, O. S., and J. C. Holm. 



1994. Oocyte recruitment in first-time spawning Atlantic 



