160 
Fishery Bulletin 107(2) 
Table 5 
Details of the autodiametric calibration relating follicle number (F ow ) to follicle diameter {D f px n) using a linear equation lnF oiu = 
ax In D f + b and polynomial equation (5) In F ow = axln D f + bxln Dj +c fitted to data collected by each institute: AZTI, Cefas, 
CSIC, and IMR for each species: Atlantic cod (Gadus morhua), European hake ( Merluccius merluccius ), Atlantic herring (Clupea 
harengus), Atlantic mackerel ( Scomber scombrus), European plaice ( Pleuronectes platessa), and redfish (deep water redfish 
[Sebastes mentella ] or golden redfish [Sebastes marinus]. 
Species 
Institute 
a 
b 
c 
N 
r 2 
Atlantic cod 
Cefas 
-3.106 
28.777 
28 
0.972 
Atlantic cod 
Cefas polynomial 
17.234 
-1.5497 
-37.864 
28 
0.986 
Atlantic cod 
IMR 
-2.700 
26.088 
47 
0.988 
European hake 
AZTI 
-2.157 
22.293 
157 
0.774 
European hake 
CSIC 
-2.196 
22.758 
245 
0.780 
Atlantic herring 
IMR 
-2.718 
26.287 
23 
0.971 
Atlantic mackerel 
Cefas 
-2.528 
25.030 
78 
0.761 
European plaice 
Cefas 
-2.910 
27.442 
150 
0.980 
Redfish 
CSIC 
-2.551 
25.040 
147 
0.948 
General (excluding European hake) 
All institutes except AZTI 
-2.750 
26.371 
475 
0.979 
reported for species like European hake or Atlantic 
mackerel maybe attributed to the ovary being packed 
with a larger, and perhaps more variable partial vol- 
ume of PVF associated with a continuous follicular 
distribution. Further analysis to determine the source 
of variation in the autodiametric calibration for fish 
with a continuous follicular frequency distribution is 
therefore considered worthwhile. Calibration data that 
included spawning Atlantic cod was best described by 
a polynomial model, although the additional term was 
not significant for the European plaice, even though the 
data included fish with hydrated follicles and POF. The 
difference may arise because European plaice produce 
fewer egg batches, about five (Urban, 1991), compared 
to between 14 and 21 in Atlantic cod (Kjesbu et al., 
1996b). Thus, residual POFs in Atlantic cod ovaries 
should take up increasingly more space in the ovary 
towards the end of the spawning season changing the 
relative partial volume taken up by residual vitellogenic 
follicles. 
An alternative to full automation is to use a semi- 
automatic analysis so that follicles that are not meas- 
ured by the automatic analysis can still be measured 
manually. In practice, the dominant fecundity follicles 
were measured in automatic mode and then other 
follicular types, such as POFs or atretic follicles, are 
manually assigned and measured accumulating the 
measurements in user defined classes. This informa- 
tion can be used for more qualitative aspects, such 
as an overview of atresia intensity or confirming fish 
are at an advanced state of maturity, and also to 
provide a means to exclude fish that have started 
spawning. Our experience shows that POFs will arise 
from a synchronous ovulation that will produce a 
cohort of POFs of similar size and shape thus mak- 
ing their identification more certain. In practice we 
keep a tally of identified POFs in a separate class 
and reject the fish from the fecundity data set to 
apply the annual egg production method if five or 
more POFs with similar structures are found. The 
hydrated cohort were split from the vitellogenic mode 
to determine the batch fecundity by inspection of the 
frequency distribution produced from the follicular 
measurements. This provides a further advantage 
for the study of batch fecundity because it is easier 
to see and separate the next batch compared to the 
traditional gravimetric method described previously 
(Hunter and Macewicz, 1985a). 
In conclusion the present study has shown that im- 
age analysis and the autodiametric method have wid- 
er application than originally reported (Thorsen and 
Kjesbu, 2001; Klibansky and Juanes, 2008). Although 
one report (Friedland et al., 2005) indicated caution 
in this respect, the range of spawning strategies and 
institutes participating in this study indicate that 
for species with a discontinuous follicular frequency 
distribution, the method is also reliable. However, the 
authors have demonstrated that a calibration should 
be done to validate the method in all new applications 
whether it involves new species, equipment, or situa- 
tion. The use of the pipette makes it possible to take 
quantitative fecundity samples in situations were ac- 
curate balances, measuring to an accuracy of 0.1 mg, 
will not function. In addition this provided a means to 
calibrate the autodiametric method for routine qual- 
ity control and substantially reducing the use of toxic 
fixative. Substantial histology costs can be avoided by 
improving the interpretation of whole mounts and the 
approach has great utility to study the fate of fecun- 
dity during the spawning season. 
