Fisheries Research 67 (2004) 13–23 Pattern of oocyte development and batch fecundity in the Mediterranean sardine Konstantinos Ganias a,∗, Stylianos Somarakis b,c, Athanassios Machias c, Athanasios Theodorou a a Laboratory of Oceanography, University of Thessaly, Fytokou Street, GR 38446, N. Ionia, Magnisia, Greece b Department of Biology, University of Patras, 26500 Patra, Greece c Institute of Marine Biology of Crete, P.O. Box 2214, GR 71003, Iraklio, Crete, Greece Received 24 March 2003; received in revised form 6 August 2003; accepted 18 August 2003 Abstract In the present study, the pattern of oocyte development was investigated in the Mediterranean sardine (Sardina pilchardus sardina) in order to examine whether non-hydrated females could be included in batch fecundity measurements. Gonad histol- ogy and frequency distributions of oocyte diameters demonstrated that the Mediterranean sardine exhibits group-synchronous type of oocyte development. The spawning batch begins to separate in size from the adjacent population of smaller oocytes at the secondary yolk globule stage and a well-developed size-hiatus is established at the tertiary yolk globule stage. The spawning batch could be clearly identified prior to hydration and batch fecundity-on-fish weight relationships did not differ significantly between hydrated females and females at the tertiary and migratory nucleus stages. Thus, apart from hydrated females, batch fecundity in the Mediterranean sardine may also be measured by the use of females at the tertiary and migratory nucleus stages. Relative fecundity was shown to be independent of body weight and its estimates during the respective peak spawning months for the Aegean Sea and Ionian Sea stocks were 360 eggs/g (December 2000) and 339 eggs/g (February 2001). © 2003 Elsevier B.V. All rights reserved. Keywords: Mediterranean sardine; Sardina pilchardus sardina; Oocyte development; Group-synchronous; Batch fecundity 1. Introduction pilchardus: Andreu and Pinto, 1957; Quintanilla and Pérez, 2000; Sardinops sagax: Claramunt and For multiple spawning fish species with indeter- Herrera, 1994; Macewicz et al., 1996; Sardinops minate annual fecundity the only useful fecundity melanostictus: Matsuura et al., 1991; Murayama measurement is the number of eggs produced in et al., 1994; Sardinops ocellata: Le Clus, 1979; a single spawning batch (batch fecundity) (Hunter Sardinella brasiliensis: Isaac-Nahum et al., 1988; et al., 1985). In all sardine and anchovy species and Engraulis mordax: Hunter et al., 1985; Engraulis subspecies studied so far (e.g. Sardina pilchardus capensis: Melo, 1994; Engraulis japonicus: Imai and Tanaka, 1994; Encrasicholina heteroloba: Wright, ∗ 1992; Encrasicholina punctifer: Maack and George, Corresponding author. Tel.: +32-421-093-053; fax: +32-421-093-054. 1999) females are multiple spawners exhibiting in- E-mail address: [email protected] (K. Ganias). determinate annual fecundity and continuous oocyte 0165-7836/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.fishres.2003.08.008 14 K. Ganias et al. / Fisheries Research 67 (2004) 13–23 size-frequency distribution, except in hydrated ovaries 2. Materials and methods where a clear size separation between the hydrated and non-hydrated oocytes is established. As a corol- Adult sardines were collected during two DEPM lary, the method of choice in assessing batch fe- (daily egg production method) surveys carried out in cundity in these species is the ‘hydrated oocyte the eastern Mediterranean during winter 2000–2001 method’ (Hunter et al., 1985). Nevertheless, due to at the respective peak spawning months for sar- the limited time course of hydration in small pelagic dine in the central Aegean Sea (December) and the fish and the aggregating behavior of spawning fe- central Ionian Sea (February) (Ganias et al., 2002; males (Hunter et al., 1985; Alheit, 1993), hydrated Anonymous, 2000). Such surveys provide fishery ovaries are often hard to obtain (e.g. Palomera and independent biomass estimates and involve the esti- Pertierra, 1993); thus, other more time-consuming mation of several biological variables of the popu- and often less accurate methods are applied (e.g. lation, like spawning frequency and batch fecundity the oocyte size-frequency method; Hunter et al., (Parker, 1985). Samples were collected on board 1985). both the commercial fishing fleet (purse seiners and In the present study, the pattern of oocyte develop- bottom trawlers) as well as the R/V “PHILIA” by ment was investigated in the Mediterranean sardine. means of an experimental pelagic trawl. A total of The latter differed from what has been described 19 commercial and 11 research samples were col- for other sardine and anchovy populations in dis- lected during the DEPM survey in the Aegean Sea playing a well-defined size-hiatus prior to oocyte (December 2000), and 21 commercial and 9 research hydration, which allowed for the precise micro- samples during the DEPM survey in the Ionian Sea scopic definition of the spawning batch since late (February 2001) (Fig. 1). Each sample consisted of a vitellogenic stages (i.e. tertiary yolk globule stage). random collection of 1.5–2 kg of sardines. Fish were Subsequently, batch fecundity of the Aegean Sea body-cavity slit and fixed in 10% neutral-buffered (December 2000) and Ionian Sea (February 2001) formalin, immediately after capture. sardine stocks was investigated by the use of females In the laboratory, 15–20 females were randomly with ovaries after the tertiary yolk globule stage of selected from each sample for histological analysis of development. the gonads. Whenever females with hydrated oocytes Fig. 1. Map of the study area, indicating the location of adult sampling stations: (᭹) commercial fleet samples; (ᮀ) research pelagic trawl samples; (a) Aegean Sea (December 2000); (b) Ionian Sea (February 2001). K. Ganias et al. / Fisheries Research 67 (2004) 13–23 15 were macroscopically being detected in a sample, all than 500 oocytes per gonad. Oocytes are rarely per- such fish were processed in an effort to collect and fectly spherical in shape, so maximal and minimal analyze as many hydrated females as possible. Each oocyte diameters were averaged to decrease variance selected female was weighed (0.1 g) and its ovaries and avoid artificially increasing the overlap between removed, weighed (0.0001 g) and re-preserved in different groups of oocytes (West, 1990). formalin. Subsequently, a piece of tissue from the Batch fecundity was estimated using methods de- center of each ovary was subjected to histological scribed in Hunter et al. (1985). Briefly, individual analysis, considering that oocyte development in batch size was measured in three subsamples of sardine ovaries does not vary with position (Pérez 40–60 mg of tissue taken from the anterior, middle et al., 1992). Tissues were dehydrated, cleared in and posterior part of the gonad. Ovulated gonads xylol, and embedded in paraffin. Sections (4–6 ␮m) were not used in the fecundity analysis. Sardine go- were cut and stained with Mayer’s hematoxylin and nads are homogenous and there are no significant Eosin Y (Clark, 1981). Histological classification of differences in the number of hydrated oocytes per the ovaries was based on the developmental stage of unit weight between left or right ovary or among the most advanced oocytes (West, 1990). Stages of different parts of the same ovary (Pérez et al., oocyte development were adopted from Murayama 1992). et al. (1994) (Table 1 and Fig. 2). Ovary-free weight was corrected for the effect of In order to accurately assess the pattern of oocyte fixation in 10% buffered formalin using the equation: development, oocyte measurements were taken in fresh weight (g) = 0.945·(weight in preservative (g)) ovaries of the consecutive vitellogenic and final mat- (r2 = 0.99, n = 10, Ganias, 2003). Data on the num- uration stages. A random sample of five females per ber of eggs per batch (Fi) and the ovary-free weight stage of development and region/season were ana- (Wi) measured for the ith fish were used to fit a linear lyzed. Oocytes from small pieces of the ovaries were model: mechanically separated and those >0.2 mm were mea- Fi = a + bWi + i sured (±0.02 mm) with an ocular micrometer under a binocular microscope. The 0.2 mm was used as a Linear models explained data satisfactorily in terms of cutoff value in order to exclude the permanent stock residual properties and coefficients of determination of primary oocytes. Measurements included more (Zar, 1999). ANCOVA models were used to test for Table 1 S. pilchardus sardina: histological characteristics of oocytes at different developmental stages (adopted from Murayama et al., 1994) Stage Histological characteristics Primary oocytes (Fig. 2(a)) At the beginning of this stage (chromatin nucleolar stage) oocytes consist of a limited cytoplasm densely stained with hematoxylin. A proportionally large and centrally located nucleus, occupies the greatest part of the oocyte. As the oocytes develop, the nucleo-cytoplasmic ratio decreases, and the nucleoli increase in number and arrange around the inner part of the germinal vesicle (perinucleolar stage) Yolk vesicle (Fig. 2(b)) Unstained ‘empty’ vacuoles (yolk vesicles) appear in the cytoplasm and then migrate to the periphery of the cytoplasm forming at first a single layer and then several peripheral rows (cortical alveoli). By the end of this stage the zona radiata appears Primary yolk globule (Fig. 2(c)) Yolk is incorporated to form small yolk globules within the peripheral ooplasm. Yolk globules extend 3/4 of the distance from the periphery to the perinuclear zone. Oil droplets begin to appear in the ooplasm Secondary yolk globule (Fig. 2(d)) Yolk globules increase in number and size, and consequently oocytes are filled with them. Oil droplets increase in number and fill the entire ooplasm Tertiary yolk globule (Fig. 2(e)) The oil droplets exist only in the periphery of the germinal vesicle Migratory nucleus (Fig. 2(f) and (g)) The germinal vesicle migrates toward the animal pole and the oil droplets begin to fuse to a single oil droplet Hydration (Fig.