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Fishery Bulletin 107(2) 
staining by washing through mesh sieves that retained 
all follicles larger than 125 pm using either 1:3 glyc- 
erine:water (McBride and Thurman, 2003), 0.9% w/v 
sodium chloride (Ramsay and Witthames, 1996), or 
clean water. Replacement of the fixative used for stor- 
age by saline or water did not affect the size of fol- 
licles during subsequent storage for 5 days at 0-5°C. 
Comparison of whole-mount method 
with histological method 
In order to study the morphology of POFs immediately 
after ovulation, and during advanced regression, ovarian 
samples were taken from trawl caught Atlantic cod taken 
from the Irish Sea (Table 1) during the spawning season. 
In some cases the fish (n = 10) were producing copious 
quantities of ovulated eggs and were expected to con- 
tain newly produced POFs created simultaneously with 
ovulation. After fixation in NBF the whole mounts were 
examined both unstained and after PAS staining to color 
both the oocyte chorion and the basement membrane 
between the granulosa and thecal layers of the follicle. 
The size frequency of the residual vitellogenic follicles 
and POFs were also measured at this time. Normal vitel- 
logenic and POFs were tentatively identified in the above 
preparations based mainly on their shape but also on 
their internal structure revealed as irregular blotches or 
shading (Hunter and Macewicz, 1985a, 1985b; Hunter et 
al., 1992). One fish was chosen from this group because 
it contained not only large POFs but also illustrated pre- 
vious spawning activity based on large numbers of small 
POFs assumed to come from previous ovulation events. 
Examples of tentatively identified follicular classes were 
removed from the whole mount and processed into PAS 
Mallory trichrome stained 2-hydroxyethyl methacrylate 
(Technovit® 7100 Kulzer GmbH, Wehrheim, Germany) 
sections (Witthames and Greer- Walker, 1995) in order 
to compare the accuracy of the identification. 
Alpha atretic follicles (Hunter and Macewicz, 1985b) 
were identified in biopsy samples taken with a Pipelle 
de Cornier® (Prodimed, Neuilly En Thelle, Picardie, 
France), a flexible, plastic tube 2.1 mm internal diam- 
eter, by endometrial suction after gonad catheterization 
(Bromley et al., 2000). These samples were taken from 
sedated captive Atlantic cod available from a separate 
study carried out at IMR during March 2004 to deter- 
mine the rate of transition from normal to advanced 
stage atretic follicles. Each biopsy sample was fixed as 
above and examined as an unstained and stained whole 
mount to compare the intensity of follicular atresia 
found in both preparations. Intensity of atresia (la) 
was defined as 
la = Ni/(Ni +Nj), (2) 
where Ni and Nj refer to alpha atretic and normal vitel- 
logenic follicles, respectively. 
The alpha atresia and more advanced beta follicular 
stages have been defined previously based on the frag- 
mentation or absence of the chorion (Witthames and 
Greer-Walker, 1995;Witthames, 2003) following previ- 
ous studies (Hunter and Macewicz, 1985b). After scor- 
ing the intensity of atresia the whole mounts were in- 
filtrated in resin and then polymerized slowly at -10°C 
over a period of 2 hours that all the follicles lay at the 
base of the mold. At least 25 to 30 sections of 5 pm were 
cut and discarded in order to take a section within 125 
to 150 pm from the base of the mold to transect all the 
follicles present in the sample. This section was stained 
by the PAS Mallory trichrome method to identify and 
count the transected follicles. 
Fecundity maturation and down regulation 
In order to study the change in fecundity during matu- 
ration D, and atresia data were taken from Atlantic 
cod sampled in the Irish and North Seas between Jan- 
uary and March during 2003 and 2004 (Table 1), and 
examined in two ways. In the first case the standing 
stock of atretic follicles (la) was measured as preva- 
lence (proportion of fish containing alpha atretic fol- 
licles) and relative intensity (/a/whole body weight g) 
as described previously (Witthames and Greer-Walker, 
1995). The atresia was determined in histological sec- 
tions stained by PAS Mallory trichrome. Secondly the 
overall impact of atresia on relative fecundity F bw = 
F/total body weight in g) during maturation was deter- 
mined by assessing the reduction of F bw in relation to 
D f as recently described (Thorsen et al., 2006). 
F bw - axLn (D f ) + b. (3) 
An additional data set (Table 1) was also available from 
an annual egg production survey of Atlantic cod, Euro- 
pean plaice, and common sole biomass (Armstrong et 
al., 2001) to assess whether down regulation also occurs 
in other species with a similar fecundity development 
process. This data set contained details of fecundity, 
fish length (cm) total, and ovarian weight (g) for each 
species and was used to calculate F bw in each case. D f 
was predicted from F ow using the ovarian weight and 
fecundity data in Equation 4 (below) adjusted to make 
F ow the independent variable. 
Autodiametric calibration 
A regression line (based on ln-transformed data) was 
established for each species and institute (Thorsen and 
Kjesbu, 2001) between D f and F ow using the following 
formula where a and b are equation constants. 
Ln F ow = a x In Df + b. (4) 
In one data set the parameters showed some degree 
of noncovariance and a second polynomial function 
(ln Df 2 ) was fitted to the data where a, b and c are 
constants: 
Ln F ow = a x \n Df + b x \n Dj + c. 
(5) 
