Sun et al.: Reproductive biology of Makaira nigricans 
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Materials and methods 
Collections from fish markets and measurements of fish 
Samples from 1001 blue marlin were collected randomly 
from September 2000 to December 2001 at the Tung- 
kang fish market in southwest Taiwan. All samples were 
caught by offshore longliners operating between 16-23°N 
latitude and 115-135°E longitude. Sex (determined from 
the macroscopic characteristics of the gonads, and from 
histological sections for small individuals), eye-to-fork 
length (EFL; the posterior margin of eye’s bony orbit 
to the distal end of the central ray of the caudal fin, 
cm), and round weight (RW, kg) were recorded for each 
fish. A tissue sample was randomly collected from the 
anterior (females) and middle (males) of either the right 
or left lobe of the gonad and immediately fixed in 10% 
buffered formalin for later oocyte measurement and 
histological analysis. Three ovary pairs were collected 
in July 2004 to evaluate the synchronicity of egg devel- 
opment within, and between, ovary pairs. The left and 
right lobes of these ovaries were each divided into ante- 
rior, central, and posterior portions, and each portion 
further divided into outer, middle, and central layers. 
A total of 54 0.05-g subsamples (3 ovaries x 2 lobes x 3 
portions x 3 layers) were collected randomly and the 
number of whole oocytes and the mean oocyte diameter 
(MOD) for each subsample was estimated. More specifi- 
cally, the MOD was estimated as the average of the dia- 
meters of the most advanced group of oocytes calculated 
with the Image-Pro Plus software (Media Cybernetics, 
Silver Spring, MD), after calibration against an optical 
micrometer. However, histological sectioning deforms 
the oocyte from its sphere-like shape, and three types 
of measurements were therefore used to obtain reliable 
results (Arocha, 2002). Early developed oocytes were 
measured at the major axis crossing the nucleus; matur- 
ing oocytes were measured across the nucleus from 
well-formed spheres; and the diameters of fully mature 
oocytes were calculated from the circumference of the 
oocyte divided by k. 
The condition factor, C, was determined for each fish 
by using the relationship 
C = (100,000 x RW) / EFL b , (1) 
where b = the slope of the length-weight relationship 
(King, 1995). 
The value for b was estimated by using linear regression 
after the data were log-transformed. The parameter b 
was not significantly different from 3 for females (£=0.53, 
df=172, P >0.05) and males (£=-0.99, df=210, P >0.05). 
The condition factor was related to the gonadosomatic 
index (GSI), which was determined as follows (Uosaki 
and Bayliff, 1999): 
Sex ratio in each month or size class was calculated as 
the ratio of the number of females to the total numbers 
of females and males. Chi-square tests were used to test 
for significant differences in sex ratio among months and 
sizes. Sex ratios were also regressed on length by using 
logistic regression (DeMartini et al., 2000). 
Reproductive activity 
Microscopic characteristics of histological sections and 
the most advanced group of oocytes were used to assign 
ovaries to stage (Hunter and Macewicz, 1985; West, 
1990; Arocha, 2002; Arocha and Barrios, 2009). For 
males, the classification of testicular development was 
based on the degree of spermatogenesis, the devel- 
opment of the vas deferens, and the composition of 
germ cells (Grier, 1981; Ratty et al., 1990; deSylva 
and Breder, 1997). Each 150-mm 2 3 preserved tissue 
sample was embedded in paraffin, sectioned at 7 pm, 
and stained with Mayer’s haematoxylin and eosin. The 
dynamics of the ovarian maturation process were evalu- 
ated by examining the modes in size-frequency distri- 
butions of whole oocytes (after Hunter and Macewicz, 
1985, Arocha, 2002; Arocha and Barrios, 2009). The 
95% confidence intervals for the proportion of oocytes of 
each diameter by ovarian maturity stage were obtained 
by a bootstrap procedure in which each pseudo data set 
was constructed by selecting whole oocytes at random 
and with replacement. Changes over time in the mean 
diameter of the most advanced group of oocytes, the 
GSI values (for individuals larger than >180 cm EFL 
to enhance temporal variation), and the composition of 
ovarian development stages were evaluated to deter- 
mine the spawning season. 
Size-at-maturity 
The proportion of mature fish of all assessed fish 
classes, defined as a maturity ogive, was developed 
from samples caught during the spawning season 
(Murua et al., 2003). Females were defined as sexu- 
ally mature if they had early yolked, advanced yolked, 
migratory nucleus, or hydrated oocytes (Hunter and 
Macewicz, 2003; Arocha and Barrios, 2009), whereas 
males were defined to be sexually mature if they had 
secondary spermatocytes, spermatids, or spermatozoa. 
The presence of postovulatory follicles (POF) in ovaries 
and spermatozoa in the vas deferens were respectively 
taken as evidence of recent spawning of females and 
males. Atresia of yolked oocytes was also noted for 
mature but reproductively inactive females. The prob- 
ability that the i th fish was mature (P-) was modeled 
with a logistic curve: 
Pi = 1 / (l + e ~^ n{19) [ < - EFL i~ EFL so V(EFL 5 q-efl 95 i] 
(3) 
GSI = (GW /EFL 3 ) x 10 4 , 
where GW = gonad weight (g). 
(2) where EFL l = the EFL of fish i; and 
EFL 50 and EFL 95 = the EFLs at which 50% and 95% of 
the assemblage reached maturity. 
