J. Mar. Biol. Ass. U.K. (2003), 83, 1327^1332 Printed in the United Kingdom
Ovarian atresia in the Mediterranean sardine, Sardina pilchardus sardina K. Ganias*$, S. SomarakisOP, C. KoutsikopoulosO, A. MachiasP and A. Theodorou* *University of Thessaly, Laboratory of Oceanography, Fytokou st. 38446, N. Ionia, Volos, Greece. O University of Patras, Department of Biology, Laboratory of Zoology, 26500 Rio, Patras, Greece. P Institute of Marine Biology of Crete (IMBC), PO Box 2214, 710 03 Iraklion, Greece. $ Corresponding author, e-mail:
[email protected]
Histological analysis was used to describe and analyse the process of ovarian atresia in sardine (Sardina pilchardus sardina) from the central Aegean Sea (eastern Mediterranean). The spawning potential of females in relation to intensity of atresia was evaluated and the rate that the ovary passed from the active to inactive condition was followed in ¢sh collected over an annual cycle. Early postspawning females, de¢ned as those with 100% alpha-atretic oocytes, occurred throughout the spawning period; they had lower gonosomatic and hepatosomatic index, but similar total length compared to reproductively active females.
INTRODUCTION Follicular atresia is an involutive process widespread in ovaries of ¢sh and other vertebrates. The study of prevalence and intensity of histological stages of the atretic follicles provides the chief criteria for identifying regressing ovaries and thus predicting the cessation of spawning in ¢sh populations (Hunter & Macewicz, 1985; Hunter & Lo, 1997). Furthermore, de¢nition of atretic states and subsequent assignment of females to di¡erent spawning states (active, inactive/immature) are of great importance for spawning biomass estimates through the Daily Egg Production Method (Hunter & Lo, 1997). Finally, assessment of the atretic condition of the ovary contributes to the estimation of crucial reproductive variables of ¢sh populations such as fecundity and length/age at ¢rst maturity. The sardine, Sardina pilchardus sardina, is one of the most important pelagic ¢sh in Mediterranean waters. Despite its economic importance, data on the reproductive biology of the species are sparse (Ganias et al., 2003). In the present paper, we describe the atretic follicles of the Mediterranean sardine using ¢sh collected in the central Aegean Sea from September 1999 to August 2000. The prevalence and intensity of atretic stages in relation to incidence of imminent or recent spawning, is used to assign females to di¡erent spawning states (active, early postspawning, late postspawning and immature). The rate that the ovary passes from the active to inactive condition is followed by the seasonal evolution of early postspawning females; the latter are compared with active females with respect to size and hepatic mass in order to examine whether these parameters are associated with ovarian inactivation.
MATERIALS AND METHODS Samples of adult sardines (N¼98) were collected on a monthly basis from coastal locations of the central Aegean Journal of the Marine Biological Association of the United Kingdom (2003)
Sea, from September 1999 to August 2000. Sampling was carried out on-board both the commercial purse seine and bottom trawl vessels as well as the RV ‘Philia’, by means of an experimental pelagic trawl. Each sample consisted of a random collection of 1.5^2 kg of sardines. Fish were body cavity slit immediately after capture, and ¢xed in 10% neutral bu¡ered formalin. In the laboratory, up to 20 females were randomly selected from each sample and measured for total length (0.1cm). Ovaries, liver and viscera were removed, gutted weights (W) were recorded (0.01g), and ovaries were preserved in 10% neutral bu¡ered formalin in order to be subjected to histological analysis. Both ovaries and liver were dried of surface moisture and weighed (0.1mg). Gonosomatic (GSI) and hepatosomatic (HSI) indices were calculated as the ratio of gonad (Wg) and liver (Wl) weight to gutted weight respectively, expressed as a percentage (GSI¼Wg/ W*100, HSI¼W1/W*100). A total of 949 ovaries were examined histologically. A piece of tissue was removed from the centre of each ovary, dehydrated, cleared in xylol, and embedded in para⁄n. Sections (4^6 mm) were cut and stained with Mayer’s haematoxylin and eosin Y. In each sectioned ovary, we recorded: (a) the maturation stage of the most advanced healthy oocytes (unyolked, yolked or hydrated oocytes); (b) the incidence of di¡erent classes of postovulatory follicles (POF-0, POF-1, or POF-2; Ganias et al., 2003); (c) the prevalence and intensity of alpha-atresia in yolked females (for de¢nitions see Kurita et al., 2003), and the prevalence of beta or subsequent atretic stages in unyolked females. Subsequently, based on criteria described by Hunter & Macewicz (1985) for northern anchovy (Engraulis mordax), ovaries were classi¢ed with respect to oocyte and atresia stages into four di¡erent atretic states: Atretic state 0, no alpha-atresia of yolked oocytes. Atretic state 1, 550% of yolked oocytes were in alphaatresia.
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Atretic state 2, 550% of yolked oocytes were in alphaatresia. Atretic state 3, no remaining yolk but beta or later atretic stages were present. The spawning potential in each atretic state was estimated by calculating the incidence of females with POFs (evidence of previous spawning) and hydrated oocytes (evidence of future spawning) within each state (Hunter & Macewicz, 1985). Subsequently, female sardines were classi¢ed into di¡erent spawning states: Active. Females capable of spawning at the time of capture or in the near future. Early postspawning. Yolked females not capable of spawning at the time of capture nor in the near future, although some may have spawned in the recent past; the mean duration of this state in northern anchovy is nine days (Hunter & Macewicz, 1985; Hunter & Lo, 1997). Late postspawning. Unyolked females that may have spawned in the distant past; in northern anchovy, an ovary in such state may have been active 16 days to over a year before capture (Hunter & Macewicz, 1985; Hunter & Lo, 1997). Immature. Females without indication of previous or future reproductive activity. Early postspawning females, i.e. reproductively inactive females with alpha-atretic oocytes, provide the best measure of ovary resorption in the population (Hunter & Macewicz, 1985; Hunter & Lo, 1997), and thus they were used to estimate the rate of inactivation of female spawners. On the other hand, late postspawning females cannot provide a reliable measure of the rate that individuals pass from the active to the inactive state, because their ovaries may retain atretic follicles for quite a long time (Hunter & Macewicz, 1985). Hence, females in late postspawning condition were pooled with immature females. Di¡erences among the monthly fractions of early postspawning females were tested using contingency tables analysis. Only females larger than the size at which 50% of ¢sh were mature (size at maturity, L50) were considered to avoid possible size-related bias because of di¡erent length distributions among the monthly samples. L50 was determined from the relationship between percentage (P) of mature ¢sh (females with yolked or hydrated oocytes) at length class (L). This relationship, widely used for maturity studies (Petrakis & Stergiou, 1997) is described by the logistic function: P¼
ev1 þv2 L 1 þ ev1 þv2 L
(1)
parametric (t-test: GSI, HSI) or nonparametric tests (Kolmogorov ^Smirnov test: total length).
RESULTS Morphology of atresia
Vitellogenic oocytes in alpha-stage atresia were easily recognizable, and a number of di¡erent phases were distinguished based on the shape and location of the zona radiata (chorion), proliferation of the follicle cells and breakdown of the yolk globules (Figure 1): Initial phase (Figure 1B). One of the ¢rst signs of atresia is the disintegration of the nucleus, which is recognized by its irregular shape and its dark-basophilic staining. Nuclear disintegration is accompanied by fragmentation of the zona radiata and hypertrophy of the follicle cells. Intermediate phase (Figure 1C). Zona radiata is highly fragmented and subsequently breaks up into segments of irregular shape. Granulosa cell layer is highly proliferated and its cells invade the cytoplasm through the ruptures of zona radiata to phagocytose yolk globules. The disintegration of the globules is more intense at the periphery of the ooplasm and is indicated by fused globules, or globules expanded and of a less regular shape. Advanced phase (Figure 1D,E). The disintegration process performed by the granulosa cells continues until all yolk globules are digested. Consequently, the atretic follicle is greatly shrunken, and the zona radiata/ooplasm ratio is increased. ‘Empty’, unstained vacuoles appear inside the ooplasm. At the end of the alpha-atretic stage the chorion remnants disperse into deeper layers and very few yolk globules are visible mainly at the centre of the oocyte (Figure 1E). After the completion of yolk digestion and chorion dissolution, follicular atresia continues with the resorption of the remaining granulosa and thecal cells. The atretic follicle is an amoeboid-shape, basophilic structure made up of invading granulosa cells surrounded by thecal cells (Figure 1F). One or more large unstained cavities may exist between the granulosa cells. An exceptional characteristic of follicular atresia in the Mediterranean sardine is the accumulation of yellow-brownish pigments (which are a typical characteristic of delta stage atresia; Hunter & Macewicz, 1985) in follicles with histological characteristics of beta stage atresia (Figure 1F). This indicates that the follicle is completely resorbed during the beta stage, without passing through gamma and delta stages. The latter two stages were not observed in the collections.
and the value L50was estimated from the expression: L50 ¼ v1 =v2
(2)
The calculations of v1, and v2 and of the 95% con¢dence intervals of the estimated value of L50 were performed following procedures described in Petrakis & Stergiou (1997). In order to examine whether early postspawning females di¡er from active females with respect to other characteristics, in addition to histological appearance of their gonads, these two groups of females were also compared with respect to GSI, HSI, and total length. Comparisons were performed on a monthly basis, using Journal of the Marine Biological Association of the United Kingdom (2003)
Incidence of spawning in atretic females
The percentages of females showing evidence of recent or imminent spawning (POFs or hydrated oocytes) with regard to atretic state and intensity of alpha atresia are summarized in Table 1. Of the yolked females without alpha-atretic oocytes (atretic state 0), 36.17% showed evidence of spawning. Sixty per cent of females in atretic state 1, had low (510%) intensity of alphaatresia. Such females, i.e. females with 510% yolked oocytes in alpha-atresia, showed increased incidence of spawning (48.68% of them had POFs in their ovaries). The spawning rate in the remaining state 1 females, i.e.
Ovarian atresia in sardine
K. Ganias et al. 1329
Figure 1. Sardina pilchardus sardina. Light microphotographs of oocytes at consecutive stages of atresia. (a) Healthy yolked oocyte; (b) initial phase of alpha-stage atresia, distinguished by the disintegration of the nucleus (N), fragmentations (F) of the zona radiata (ZR), and slight proliferation of granulosa cells (GCL); (c) intermediate phase of alpha-stage atresia, with zona radiata (ZR) fragmented but in position, and granulosa cells (GCL) highly proliferated; (d) advanced phase of alpha-stage atresia distinguished by an increase in the chorionic remnants (ZR)/ooplasm ratio, and the appearance of ‘empty’, unstained vacuoles (V); arrows: fused globules; (e) advanced phase of alpha-stage atresia, with chorionic remnants dispersed into deeper layers and very few visible yolk globules (arrows); (f) atretic follicle with characteristics of beta-atresia [e.g. intercellular cavities (c) and thecal cell layer (T)] containing yellow-brownish pigments (P). Scale bar, 0.1 mm.
Figure 2. Sardina pilchardus sardina. Light microphotographs of ovaries at di¡erent spawning states. (a) Active ovary containing yolked oocytes, alpha-atretic oocytes (a), and postovulatory follicles (POFs); (b) early postspawning ovary with 100% yolked oocytes in alpha-atresia (a); (c) late postspawning ovary indicated by the co-occurrence of primary oocytes and beta atretic follicles (b); (d) immature ovary.
in females with 10^50% alpha-atresia, was similar to that of females in atretic state 0 (37.74% of them showed evidence of spawning). Two groups were recognized among females with state 2 atresia. In the ¢rst group, females had 50^70% of their Journal of the Marine Biological Association of the United Kingdom (2003)
vitellogenic oocytes in alpha-atresia. In the second group, females had 100% of their vitellogenic oocytes in alphaatresia. The percentage of females in the ¢rst group showing evidence of spawning was lower (11%) than that recorded for females in atretic states 0 and 1. However,
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Table 1. Sardina pilchardus sardina. Assignment of females into spawning states: number of females in di¡erent histological classes with regard to atretic state (sensu Hunter & Macewicz, 1985) and intensity of alpha atresia (% of a¡ected follicles). S, Per cent of females with evidence of imminent or recent spawning (with hydrated oocytes or postovulatory follicles [POFs]). Histological class Postovulatory follicles Atretic alpha-atresia state (%) 0 1 2
Hydrated oocytes
0 510 10^50 50^70 100
5
POF-0
POF-1
POF-2
74 21 11 2
50 6 4 3
37 10 5 1
No hydrated oocytes /POFs
S (%)
Spawning state
293 39 33 50 62 25
36.17 48.68 37.74 10.71 0.00 0.00
Active Active Active Active Early postspawning Late postspawning
3
Table 2. Monthly fractions of active (A), early-postspawning (EP), and late postspawning/immature sardines (LP/I) and respective averages of gonosomatic index (GSI), hepatosomatic index (HSI), and total length for active and early-postspawning females for the study period. t, statistic of the t-test; k, statistic of the Kolmogorov^Smirnov test. Statistical tests were performed only when both groups of females consisted of more than ¢ve ¢sh. SE in parentheses. Fractions Year
Month
1999
2000
GSI
HSI
A
EP
LP/I
A
EP
Sep Oct
0.00 0.24
0.00 0.02
1.00 0.73
Nov
0.92
0.03
0.05
Dec
0.93
0.04
0.03
Jan
0.88
0.04
0.08
Feb
0.68
0.23
0.09
Mar
0.76
0.13
0.11
Apr
0.67
0.18
0.16
May
0.38
0.25
0.38
Jun
0.25
0.23
0.52
Jul Aug
0.00 0.00
0.00 0.00
1.00 1.00
2.26 (0.25) 3.71 (0.12) 3.79 (0.11) 4.36 (0.23) 4.18 (0.25) 3.39 (0.23) 2.77 (0.31) 1.36 (0.59) 2.96 (0.50)
0.66 (0.03) 1.56 (0.51) 1.20 (0.10) 2.89 (0.15) 1.60 (0.18) 1.23 (0.20) 1.32 (0.17) 0.81 (0.06) 1.35 (0.20)
3.71 (0.07)
1.44 10.27** (0.11)
All months
t
3.28** 5.31**
5.78** 3.83** 2.36*
A
EP
Length t
A
EP
1.06 (0.06) 1.38 (0.03) 1.42 (0.03) 1.33 (0.05) 1.83 (0.07) 2.28 (0.06) 2.08 (0.08) 2.07 (0.35) 2.90 (0.12)
0.62 (0.07) 1.04 2.28* (0.25) 0.81 4.71** (0..08) 0.90 (0.04) 1.38 3.30** (0.07) 1.89 2.41* (0.17) 1.76 2.02* (0.11) 1.67 (0.32) 2.38 (0.21)
14.38 14.50 (0.22) (0.30) 13.84 14.18 (0.09) (0.27) 13.98 14.15 (0.07) (0.54) 14.25 13.67 (0.13) (0.78) 13.33 13.15 (0.12) (0.22) 14.36 13.85 (0.17) (0.34) 14.39 14.30 (0.27) (0.45) 15.30 16.65 (1.03) (0.05) 13.99 (13.87 (0.16) (0.32)
1.56 (0.02)
1.38 (0.07)
13.97 (0.05)
2.44*
13.87 (0.16)
k
0.40 0.25
0.23 0.33 0.42
0.15
*, P50.05; **, P50.01.
despite their decreased spawning rate, such females were still reproductively active. On the contrary, none of females with 100% alpha-atretic follicles showed evidence of spawning, and thus, such females were considered as early postspawning. Consequently, the rate at which females passed from the active to the inactive state was measured by the use of females with 100% alpha-atretic follicles. The characteristic histological appearance of females in di¡erent spawning states is exempli¢ed in Figure 2. Journal of the Marine Biological Association of the United Kingdom (2003)
Seasonal occurrence and characteristics of early postspawning females
L50 was estimated to be 11.84 cm (95% con¢dence interval: 11.65^12.03). Early postspawning females, i.e. females with 100% alpha-atretic follicles, were not only present at the last months of the reproductive period (May ^ June), but occurred throughout the spawning season. During peak spawning months (November ^ January) the fraction of early postspawning females was low (2.5^4.4%) but constant (w2 ¼1.01, P40.05) (Table 2).
Ovarian atresia in sardine In February, the fraction increased (22.7%) and remained relatively constant until June (w2 ¼5.73, P40.05). In all months, early postspawning females had lower HSI and GSI values compared to active females (Table 2). On the other hand early postspawning females and active females did not di¡er signi¢cantly with respect to total length in any month of the study (Table 2).
DISCUSSION The microscopic examination of the atretic process in the ovaries of the Mediterranean sardine revealed similar morphological features to those described for northern anchovy by Hunter & Macewicz (1985). First, the yolked atretic oocyte undergoes full yolk resorption (alpha-stage atresia) and, secondly, the remaining follicle is completely resorbed. However, compared to northern anchovy, the atretic process in the Mediterranean sardine displays two exceptional features. The ¢rst involves the evolution of the shape/location of chorion (zona radiata), which remains in position only in early alpha-atresia; in later phases, large chorionic fragments move into deeper layers of the atretic oocyte and do not completely dissolve until all yolk is digested. A similar pattern has also been described in herring, Clupea harengus, (Kurita et al., 2003). The second exceptional characteristic involves the accumulation of yellow-brownish pigments [which consist of a typical characteristic of delta stage atresia (Hunter & Macewicz, 1985)] in follicles with histological characteristics of beta stage atresia. The latter indicates that the follicle is completely resorbed during the beta stage, without passing through gamma and delta stages. State 1 atretic females did not exhibit lower spawning rate compared to yolked females without alpha-atresia (state 0 atresia). This is opposed to northern anchovy, where the spawning rate of females classed in atretic state 1 is less than half of that of yolked females without ovarian atresia (Hunter & Macewicz, 1985). Therefore, we may conclude that low atretic losses are a regular phenomenon among actively spawning sardines, which should not be associated with the decline of the spawning rate and the cessation of spawning. Low atretic losses at peak spawning months are also reported for the Iberian sardine (Sardina pilchardus pilchardus) by Zwolinski et al. (2001), who have noticed a high prevalence of females with low intensity of atresia. However, attention should be given to the issue of whether or not such atretic losses account for di¡erences in fecundity, and therefore a¡ect the spawning potential of the population. Higher intensity of alpha-atretic oocytes (50^70%) was shown to be associated with a reduced spawning rate, though such females still showed evidence of spawning, which implied that they were still reproductively active. Hence, we may conclude that, in the Mediterranean sardine, all females that contain a population of healthy oocytes may be considered as reproductively active. In our collections, the population of una¡ected oocytes was never less than 30% of the entire population of yolked oocytes (healthy and atretic). The inactivation of the ovary in the Mediterranean sardine seemed to be generated by mass atretic events, as, among yolked females, only those with 100% alpha-atretic vitellogenic oocytes did not show any evidence of previous or imminent Journal of the Marine Biological Association of the United Kingdom (2003)
K. Ganias et al. 1331
spawning. For this reason, the latter females were considered to be the early postspawning state, and were subsequently used to follow the rate of ovarian regression. In other populations of multiple spawners, like the northern anchovy (Hunter & Macewicz, 1985), the Iberian sardine (Pe¤rez & Figueiredo, 1992) and the European horse mackerel (Karlou-Riga & Economidis, 1996), the cessation of spawning was monitored by the use of females with 450% of alpha-atretic oocytes. The exceptional pattern of oocyte development in the Mediterranean sardine might help to explain the 100% intensity of atresia in early postspawning female sardines. According to Ganias et al. (in press), the Mediterranean sardine displays a particularly low rate of clutch recruitment; e.g. in most cases, when oocytes of the advanced batch are in some stage of true vitellogenesis, oocytes of the subsequent batch have not yet recruited to the yolked stage. Therefore, supposing that, at ovarian inactivation, the batch that would be spawned is ¢rstly and totally a¡ected by atresia, such ovaries would contain 100% alpha-atretic oocytes. On the other hand, in populations with higher rates of clutch recruitment, recruiting oocytes, which follow the advanced batch of oocytes, may also be vitellogenic. In the same sense, i.e. supposing that atresia is batch speci¢c and the advanced batch is ¢rstly and totally a¡ected by atresia at ovarian inactivation, early postspawning females may contain a totally atretic advanced batch, and a population of healthy, yolked, recruiting oocytes, not yet started to be resorbed. Early postspawning female sardines occurred throughout the population spawning period (October ^ June), even at peak spawning months. These results seem counterintuitive, since inactivated spawners are expected to occur only at the end of the reproductive period (Hunter & Lo, 1997). Macewicz et al. (1996) report a similar pattern for the population of the paci¢c sardine, Sardinops sagax, and speculate that this may be caused by the inclusion of subpopulations having di¡erent spawning seasons. Alternatively, we hypothesize that in such populations, individuals do not enter/leave the spawning stock simultaneously, and that the inactivation of spawning ¢sh is a continuous event, which peaks during the last months of the reproductive period. During all spawning months, early postspawning females had similar lengths to active spawners. Therefore, while the onset of spawning depends primarily on the size of the individual, i.e. the acquisition of size at ¢rst maturity by recruit spawners, cessation of spawning in the Mediterranean sardine did not seem to be size related, i.e. smaller females did not abandon the spawning stock earlier compared to larger females. Similar pattern has also been described for the anchovy, Engraulis ecransicolus, by Milla¤n (1999); whereas larger females began to reproduce earlier in the season than small individuals, the end of reproductive period was synchronized for all size-classes. On the other hand, early postspawning females di¡ered from reproductively active females with respect to ovarian and hepatic mass. The former exhibited lower GSI, which is a typical characteristic of the regressing ovary. They also exhibited a lower HSI, suggesting that the cessation of spawning might be be related to the exhaustion of body/hepatic energy reserves.
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We thank Dr C. Papaconstantinou, E. Caragitsou and A. Siapatis from the National Centre for Marine Research in Athens for their collaboration in the collection of the purse seine samples. Dr G. Koumoundouros and Dr N. Papandroulakis are thanked for their assistance in microscopic photography and M. Papadaki for her help in laboratory analysis. The comments of two anonymous referees are much appreciated. This research was funded by an EU Research Project, DG XIV, Contract no. 98/0039. This study represents part of the PhD dissertation of K. Ganias submitted to the Department of Agriculture, Animal Production and Marine Environment, University of Thessaly, Greece.
REFERENCES Ganias, K., Somarakis, S., Machias, A. & Theodorou, A., in press. Pattern of oocyte development and batch fecundity in the Mediterranean sardine. Fisheries Research. Ganias, K., Somarakis, S., Machias, A. & Theodorou, A., 2003. Evaluation of spawning frequency in a Mediterranean sardine population (Sardina pilchardus sardina). Marine Biology, 142, 1169^1179. Hunter, J.R. & Lo, N.C.H., 1997. The Daily Egg Production Method of biomass estimation: some problems and potential improvements. Ozeanogra¢ka, 2, 41^69. Hunter, J.R. & Macewicz, B., 1985. Rates of atresia in the ovary of captive and wild northern anchovy, Engraulis mordax. Fishery Bulletin, 83, 119^136.
Journal of the Marine Biological Association of the United Kingdom (2003)
Karlou-Riga, C. & Economidis, P., 1996. Ovarian atretic rates and sexual maturity of European horse mackerel, Trachurus trachurus (L.) in the Saronikos gulf (Greece). Fishery Bulletin, 94, 66^76. Kurita, Y., Meier, S. & Kjesbu, O.S., 2003. Oocyte growth and fecundity regulation by atresia of Atlantic herring (Clupea harengus) in relation to body condition throughout the maturation cycle. Journal of Sea Research, 49, 203^219. Macewicz, B., Castro Gonzalez, J.J., Cotero Altamirano, C.E. & Hunter, J.R., 1996. Adult reproductive parameters of Paci¢c sardine (Sardinops sagax) during 1994. California Cooperative Oceanic Fisheries Investigations Report, 37, 140^151. Milla¤n, M., 1999. Reproductive characteristics and condition status of anchovy Engraulis ecransicolus L. from the bay of Cadiz (SW Spain). Fisheries Research, 41, 73^86. Pe¤rez, N. & Figueiredo, I., 1992. First approach to the study of atresia in the ovary of sardine, Sardina pilchardus. Bolet|¤ n del Instituto Espan‹ol de Oceanograf|¤a, 8, 191^199. Petrakis, G. & Stergiou, K.I., 1997. Size selectivity of diamond and square mesh codends for four commercial Mediterranean ¢sh species. ICES Journal of Marine Science, 54, 13^23. Zwolinski, J., Stratoudakis, Y. & Soares, E., 2001. Intra-annual variation in the batch fecundity of sardine o¡ Portugal. Journal of Fish Biology, 58, 1633^1645. Submitted 27 May 2003. Accepted 29 October 2003.
