Witthames et a I.: Advances in methods for determining fecundity in marine fishes 
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Table 3 
Comparison of mean (standard error [SE]) follicle diameter (33^ pm), number of follicles per gram ovary (F 0UI ), circularity, and 
fecundity (F * ovary mass [g]), found in samples taken with either a pipette (P, from fresh tissue) or gravimetric method (G, from 
fixed tissue) from fish caught in the Irish Sea by commercial vessel during 2007. In each case five replicates were taken from a 
central location of the same ovary in ripe mature Atlantic cod (Gadus morhua, cod m), hydrated Atlantic cod (cod h), ripe mature 
Atlantic haddock (Melanogrammus aeglefinus, had), and European plaice ( Pleuronectes platessa, pie). The mass (g) of the ovary 
fresh (ovary F) and after storage in fixative for longer than 14 days (ovary S) is also shown. 
Parameter 
Sample 
method 
Cod m (n= 5) 
Cod h (ti = 5) 
Had (n = 5) 
Pie (72 = 5) 
Paired 7-test 
77 = 15 P 
D f 
P 
797(3) 
944(5) 
571 (4) 
1081 (10) 
0.0026 
G 
777 (3) 
904(8) 
560(1) 
1085(7) 
F ow 
P 
3613 (90) 
1460(338) 
9355 (338) 
1196(38) 
6.652- 10 
G 
3975 (149) 
1706(33) 
10394(149) 
1280(17) 
Circularity 
P 
0.986(0.001) 
0.995 (0.001) 
0.985 (0.001) 
0.990 (0.001) 
0.0015 
G 
0.966(0.004) 
0.983(0.001) 
0.961 (0.002) 
0.977 (0.002) 
Fecundity 
P 
419,132 
388,460 
205,804 
44,260 
G 
443,191 
421,424 
218,664 
45.980 
Ovary 
F 
116.0 
266.0 
22.0 
37.0 
Ovary 
S 
111.5 
247.0 
21.0 
35.9 
Fecundity (F) was calculated from the product of ovarian 
weight ( O ) and F ow . 
Statistics 
Regression analysis was carried out using the “R”(vers. 
2.5.0, Free Software Foundation, Boston, MA) and 
residuals were plotted to check there was no systematic 
pattern suggesting that the models should be further 
refined. The coefficient of variation (CV) was determined 
for predictions with new data to examine the precision 
of the fecundity estimate for a range of follicular sizes 
typical for each species. 
Results 
Ovarian sampling, follicle measurement equipment, 
and homogeneity 
In the course of more extensive use at sea, the pipettes 
performed well taking samples from maturing ovaries 
providing that they contained vitellogenic follicles vis- 
ible to the unaided eye (>400 pm). However, ovaries that 
were close to being spent or immature did not yield quan- 
titative samples because the connective tissue attached 
to the follicles pulled the sample out of the pipette as it 
was withdrawn from the ovary. When this occurred it 
was clear that the glass tube was only partially filled 
and the sampling process could be repeated to fill the 
pipette to avoid under sampling although this was not 
always successful. In summary we found that replicate 
subsamples of 25 and 100 pL tissue taken with the 
pipette from a Atlantic cod ovary equated to a gravi- 
metric sample of 26.0 mg (CV=1.8%, 72=10) and 106.0 mg 
(CV=3.7%, 72=10), respectively. 
Compared to fixing the ovary whole for the gravimet- 
ric method, the pipette procedure for collecting ovar- 
ian subsamples was found to significantly increase 
(P=0.003, P<0.0001, and P=0.002) Dp circularity of fol- 
licles (Table 3), and decrease F ow , respectively. Ovarian 
weight after fixation over 63-65 days showed a small 
decease (95% SE = 0.9) that was not apparently related 
(P= 0.55, n = 4) to the amount of NBF used to fix the 
ovary. After accounting for a reduction in ovarian mass 
the overall reduction in fecundity, determined from the 
pipette samples, was 5.7% (SE = 0.3) less compared to 
gravimetric samples taken from the same ovary fixed 
whole. 
There was a very significant difference in F ow , Dp 
and Lc means (P<0.001) between fish but there was no 
consistent trend either between the pair of ovaries or 
within the ovary at three sites (anterior, middle, and 
posterior) where samples were taken (Fig. 1). In two out 
of the seven fish there was a site effect (left, posterior, 
and right middle) on D f and Lc, but their rank order 
was reversed at other locations. It was also noticed that 
in more mature Atlantic cod ovaries, where the Lc was 
larger, the CV of Dp and Lc amongst replicates also 
increased. Similarly, sampling site (anterior, middle, 
or posterior part of one ovary) in 103 European hake 
indicated that F n , , either classified by cortical alveoli, 
early, or late vitellegenic follicle development stages, or 
all classes combined, was not related to ovarian position 
(P=0.133, 0.149, 0.789, 0.101, respectively). 
Stain evaluation 
Compared to unstained follicles the use of each stain to 
color European hake, Atlantic cod, Atlantic mackerel, 
and European plaice follicles increased the efficiency of 
image analysis measurement, particularly of semitrans- 
