Klibansky and Juanes; Effects of preservation on oocytes of tfiree Atlantic fish species 



545 



oocytes were measured. During that time, 

 swelling that was inhibited by the dense 

 packing of oocytes in the ovarian lobe may 

 have occurred; therefore by the time they 

 were measured, oocytes were very similar in 

 size to those preserved in the sub-formalin 

 treatment. 



Standard Gilson's solution has been a com- 

 mon oocyte preservative since it was devel- 

 oped. Joseph (1963) found that tuna oocytes 

 preserved in standard Gilson's solution were 

 24% smaller than those preserved in 4% 

 formalin. In contrast Schaefer and Orange 

 (1956J concluded that mean diameters of oo- 

 cytes from the same tuna species, preserved 

 in standard Gilson's solution and formalin 

 (concentration not specified) were similar, 

 although they did not present statistical evi- 

 dence to support this conclusion. Cod, had- 

 dock, and plaice oocytes preserved in modi- 

 fied Gilson's solution were 13.28%, 13.77%, 

 and 18.43% smaller, respectively, than those 

 preserved in 10% formalin. These results 

 were all significant, and are similar to Jo- 

 seph's (1963) results. The discrepancy be- 

 tween our results and those of Joseph (1963) 

 could be attributed to interspecies variation. 

 Although differences were identified, we con- 

 sider the effects of modified Gilson's solution 

 to be fairly consistent among species. 



Ethanol is rarely used to preserve ovar- 

 ian material used in fecundity studies, but 

 because Black and Dodson (2003) used etha- 

 nol to successfully fix and preserve water 

 fleas iDapluiia pulex) and their eggs, and 

 ethanol caused less distortion and change 

 in body size than a solution of sucrose and 

 4% formalin, it was evaluated in this study. 

 Oocytes preserved in this treatment were 

 of sufficient quality for use in digital im- 

 age analysis. Ethanol also had a bleaching 

 effect on oocytes, which resulted in great- 

 er contrast between oocytes and the black 

 background. The ethanol treatment tended 

 to cause smaller changes to oocyte size and ovarian 

 material weight than other treatments, except for the 

 large increase in subsample weight in plaice. In etha- 

 nol, %4(y, was negative for cod, %^od ^^^ negative for 

 cod and haddock, and both were positive for plaice. Such 

 results make it difficult to generalize about the effects 

 of ethanol on ovarian material across taxa and may 

 make this treatment less desirable than others. 



Freezing is one of the most common methods of ovary 

 preservation, but this treatment has not been used to 

 preserve oocytes for digital image analysis. Although 

 freezing was expected to result in many ruptured oo- 

 cytes, oocytes in the frozen treatment maintained a 

 very round shape and rarely broke even after vigorous 

 shaking and agitation. Freezing the oocytes solid in 

 distilled water may have improved the ultimate quality 



60—1 



Atlantic cod, combined 



Atlantic cod, Georges Bank 



Atlantic cod, Gulf of Maine 



□ Haddock, Georges Bank 



American plaice. 

 Gulf of Maine 



Gilson's Ettianol Freezing 



Treatment 



Split- 

 Formalin 



Figure 2 



Percent difference in mean oocyte diameter (%4g£,) for each species 

 (Atlantic cod [Gadus morhua], haddock [Melanogrammus aegle- 

 finus], and American plaice [Hippoglossoides platessoides]) and 

 region, grouped by treatment. For Atlantic cod, observations are 

 displayed together in the combined group and are then displayed 

 separately by region (i.e., Georges Bank or the Gulf of Maine). 

 The horizontal line at zero on the vertical axis indicates values 

 where the '^cAqj^ between experimental treatments and the control 

 treatment (formalin) was equal to zero. Circles indicate outliers. 

 Asterisks indicate mean %4o£, was significantly different from 

 zero at the appropriate Bonferroni adjusted a-value. Box length is 

 equal to the interquartile range containing the central 50% of the 

 observations (257f above and below the median). Whiskers extend 

 1.5 box-lengths above and below the box. 



of the samples by limiting the exposure of oocytes to the 

 oxidizing and desiccating effects of air. Although the 

 quality of the images obtained from these samples was 

 high enough for measurement and counting, percent 

 change in ovarian material weight and oocyte diameter 

 of frozen specimens proved to be very variable and often 

 very high (Figs. 1 and 2). 



Although the mean diameter of cod, haddock, and 

 plaice oocytes preserved in the freezing treatment was 

 23.29%, 28.88'7(, and 9.90% larger, respectively, than 

 the mean diameter of oocytes in the formalin treatment, 

 Ramon and Bartoo (1997) found that the mean diameter 

 of tuna eggs in 10% buffered formalin was generally 

 5-10% larger than the mean diameter of oocytes from 

 frozen ovaries. It may not be surprising to find a differ- 

 ence between the species we studied and the tunas, but 



