Abstract: The present study was carried out to identify the developmental changes in the gonads of Scomberoides lysan during maturation in order to determine the spawning pattern, ovulation pattern and spawning season. S. lysan is one of the most economically important Carangid food fish in Sri Lanka. Samples were obtained from June 2010 to May 2012 from the Sri Lankan waters and a total number of 570 ovaries and 525 testes were analyzed macroscopically and microscopically. Oocyte diameters of selected samples were measured in order to find out the ovulation pattern. Histologically ovaries were categorized in to seven stages by identifying chromatin nucleolus, peri nucleolus, cortical alveolar, yolk globular, late yolk, hydrated, atresia and post ovulatory follicle stage oocytes and testes were categorized in to four stages. Oocyte diameter range from 12.5-450.0 μm. Occurrence of several batches of oocytes at a time and the presence of different type of oocyte in an ovary revealed that this would be a multiple spawner with group synchronous ovulation pattern. Hydrated and post ovulatory follicle stage oocytes and spawning stage testes were only available during June and September. Immature stages of both gonads were available throughout the study period. Observations on seasonal maturity stages indicate that this species is a multiple spawner with two peak spawning season in June and September. The results obtained from the present study can be used in the management of S. lysan in the Sri Lankan waters to ensure the sustainable utilization of this species.
INTRODUCTION
Categorizing the ovaries into developmental stages and measuring the oocyte diameter distribution using histological staging system will provide detailed information on ovulation pattern, spawning season and abundance of maturity stages. Oocyte growth and development are the important issues in the reproductive biological studies of fishes (Tyler and Sumpter, 1996). Mackie and Lewis (2001) explained that the most accurate and detailed means of staging of gonads is by microscopic examination of histologically prepared sections of each specimen. Ovarian development usually defines the spawning season and number of offspring produced during spawning (De Martini and Fountain, 1981).
Histological studies are important to invent new and effective methods for increasing efficiency of brood stock, increasing fish production and ultimately increase efficiency and higher fish are predicted. Determining the peak period of spawning, exploitation level, understanding the biological characteristics and life cycle of a species are important in the management and reconstruction of a fish species (Saeed et al., 2010). Stahl and Kruse (2008) stated that classification of ovaries into developmental stages is a prerequisite for setting annual catch quotas using a harvest rate strategy based on spawning biomass estimates. Information on maturation and spawning of species will contribute to the knowledge of their population dynamics and management of the stocks (Gabr et al., 1998).
Scomberoides lysan is commonly known as double spotted queen fish belongs to the family Carangidae, distributed through the Indo-West Pacific region (Froese and Pauly, 2012; De Bruin et al., 1994) and play a major role in the ecosystem as carnivore particularly piscivores in their adult stage and economically important as food fishes more popular as in the form of dried fish (Thulasitha and Sivashanthini, 2012; Anonymus, 2008) as well as popular game fishes (Honebrink, 2000).
Various studies have been made on reproductive biology of fish and fish related organisms from different parts of the world. Few examples for such reproductive biology studies are by Chelemal et al. (2009) for Liza abu, Karatas and Sezer (2005) for Cyprinus carpio, Sivashanthini et al. (2008) for Gerres abbreviatus, Azadi and Mamun (2004) for Amblypharyngodon mola, Ismen et al. (2004) for Balistes capriscus, Shinkafi and Daneji (2011) for Synodontis eupterus and Fernando et al. (2006) for Amphiprion sebae. From the literature survey it is apparent that the reproductive biology based on histological studies of Scomberoides lysan have not been studied, so far. The present study was carried out to identify the ovulation and spawning pattern through microscopic stages of gonads of Scomberoides lysan for the first time.
MATERIALS AND METHODS
Fish samples were collected weekly between June 2010 and May 2012 from commercial catches of Jaffna, Trincomalee, Mannar and Puttalam fish landing centers in Sri Lankan waters located in the Indian Ocean (between 79-80°E longitudes and 9-10°N latitudes). Fresh samples were brought to the laboratory and gonads were obtained by the dissection. Macroscopic staging of gonads were assigned as maturity stages according to Mackie and Lewis (2001) and West (1990).
Sub sample of four specimens (2 male and 2 female) from each 10 cm total length size range was taken from each monthly sample and gonads of each specimen were taken for histological inspection.
For histological examination different portions of ovaries and testis were fixed in formal saline (8.5 g of sodium chloride, 100 mL of 40% Formalin and 900 mL of distilled water). Clearing and paraffin embedding were performed using standard histological techniques (Clark, 1981; Ratcliffe, 1982) after one week they were dehydrated in graded alcohol series, exposed to Xylene and embedded in paraffin wax. Sections of 6 μm thickness were prepared and stained with Harris Haematoxylin and Eosin, then mounted with DPX. Photographs were taken from the OPTIKA binocular light microscope equipped with AIPTEK-AHD Z600 camera. Microscopic staging of gonads were assigned in to different maturity stages according to Mackie and Lewis (2001) and West (1990).
Oocyte diameter of all maturity stages were measured for key samples using ocular micrometer and the percentage occurrence were plotted against the oocyte diameter class interval in order to confirm the ovulation pattern.
RESULTS
A total number of 570 ovaries and 525 testes were examined. Developmental stages of ovaries were categorized into seven stages as immature, maturing, rest/developing, mature, pre spawning, spawning and spent and males were categorized in to four stages as immature, maturing, mature and spawning according to their macroscopic and microscopic features.
Among the total catches of females, immature stage ovaries (60%) were dominated and obtained throughout the study period. Maturing stages were obtained from February to August (15.5%) whereas mature stages (3%) during August and October. Pre spawning and spawning (20.5%) were only available during June and September. Only <1% of spent stages with post ovulatory follicles were collected during September. Less than 1% of rest developing or second time developing stages was available during September 2011, only.
Ovarian developmental stages
Stage I: Immature ovary
Macroscopic features: Small, thread like ovaries with pink and translucent
colour; without visible oocytes (Fig. 1). It is difficult
to determine the sex morphologically in the earlier stage.
Microscopic features: Two stages were identified such as chromatin nucleolar stage (Fig. 1, 2a) and perinucleolar stage (Fig. 2a, b). Chromatin Nucleolar Stage (CNS) is characterized by large spherical nucleus which occupies the greater portion of oocyte and strongly basophilic smooth cytoplasm appeared as dark purple in colour. CNS stage oocytes grow up to 50 μm. In the later stage they develop in to perinucleolus stage (PNS) which is characterized by the appearance of several nucleoli in the peripheral region of oval shaped nucleus. Cytoplasm is still strongly basophilic in nature and these stage oocytes reached to 100 μm. Small spindle shaped follicular cells starts to proliferate and surrounds the oocytes. Weakly eosinophilic layer (precursor of zona radiata) also starts to appear between the follicular cells and oocytes. But in the later stage, large PNS oocyte may develop small vacuoles in the cytoplasm, very close to the nucleus.
| Fig. 1(a-b): | Stage I: Immature ovary view; (a) Whole view and (b) Transverse section, CNS: Chromatin nucleolus, Bv: Blood vessel, T: Tunica, La: Lamella, Total length of fish is 24 cm |
| Fig. 2(a-b): | Stage I: nucleolus stages in immature ovary, (a) Chromatin nucleolus and (b) Peri-nucleolus, CNS: Chromatin nucleolus stage, PNS: Peri-nucleolus stage, O: Oogonia, n: Nucleolus, F: Follicular cells |
| Fig. 3(a-b): | Stage II: maturing ovary view; (a) Whole view and (b) Cortical alveoli stage, PNS: Peri-nucleolus stage, N: Nucleus, F: Follicular cells, O: Oil droplets, Va: Vacuoles, Total length of fish is 48 |
Stage II: Maturing ovary
Macroscopic features: Medium size ovaries usually appeared as translucent
pink, flattened, flaccid and relatively inconspicuous. Oocytes are not visible
through the ovarian wall. External surface is smooth and uniform in appearance
(Fig. 3a).
Microscopic features: The Cortical Alveoli Stage (CAS) in which yolk deposition initiated. In this stage, cytoplasm becomes weekly basophilic nature and the nucleus is about half of the oocyte and still occupies a central position and it contains several nucleoli; Small clear staining yolk vesicles appear throughout the mid and outer regions of the cytoplasm and forming a narrow row (cortical alveoli) near the periphery of the cytoplasm and clear staining oil droplets also appear within the inner region of the cytoplasm, increasing in number and size around the central nucleus. Follicular cells become enlarge and surrounds the oocytes. This stage of ovary also contains CNS and PNS oocytes. Large oocytes grow up to 150 μm (Fig. 3b).
Stage III: Rest/developing ovary
Macroscopic features: It is difficult to differentiate macroscopically
with first time maturing stage. Colour is typically semi-translucent rose/purple;
the ovarian wall and blood capillaries are thick. But the lumen is large when
made a transverse section. Few oocytes can be seen in the late stage (Fig.
4a).
Microscopic features: Second time developing ovary with several stages of oocytes such as CNS, PNS and CAS but the dominating stage is depending on the season; Lamellae very thin; ovarian lumen is larger than fist time developing ovary. Some large oocytes reached to 200 μm (Fig. 4b).
Stage IV: Matured ovary
Macroscopic features: Large, rounded, yellow to orange coloured ovaries,
occupying about 75% to almost filling body cavity. Ovarian wall is thin and
transparent. Small opaque oocytes can be seen clearly through the ovarian wall.
Prominent blood capillaries also formed (Fig. 5a).
Microscopic features: This stage starts with early YGS. In the early stage ovary is dominated by early YGS and Previtellogenic oocytes (PVO). YGS oocytes mature and grow, causing the lamellae to expand the lumen to decrease and the tunica to stretch and thin. Vascular tissue becomes more common. In the later stage (Yolk globule stage/advanced vitellogenic oocytes) yolk granules almost cover the cytoplasm; nucleus starts to migrate towards the periphery (also refereed as migratory nucleus stage-MNS) of the oocytes. In the ovary, 300-350 μm size oocytes dominate but few of them reached to 450 μm. This stage of ovary also consists of CNS, PNS and CAS oocytes (Fig. 5b).
| Fig. 4(a-b): | Stage III: rest/developing (maturing) ovary view; (a) Whole view and (b) Cortical alveoli stage, CAS: Cortical alveoli stage, PNS: Peri-nucleolus stage, CNS: Chromatin nucleolus, Lu: Lumen, La: Lamella, Total length of fish is 71 cm |
| Fig. 5(a-b): | Stage IV: matured ovary view; (a) Whole view and (b) Yolk globule stage oocyte, Yg: Yolk globule, Mn: Migratory nucleus, F: Follicular cells, Zr: Zona radiata, Total length of fish is 68 cm |
| Fig. 6(a-b): | Stage V: pre-spawning stage ovary, (a) A portion of pre spawning ovary and (b) Late yolk stage oocyte, Mn: Migratory nucleus, Yg: Yolk globules, O: Oil droplets, Zr: Zona radiata, F: Follicular cells, Total length of fish is 78 cm |
Stage V: Pre spawning ovary
Macroscopic features: It is difficult to differentiate pre spawning stage
from the stage VI-spawning stage ovary by macroscopic analysis (Fig.
6a).
Microscopic features: Large oil droplets increase in size and intermixed with the yolk globules. Nucleus starts to migrate towards the periphery of the oocyte. Hydrated oocytes and post ovulatory follicles can be seen if the spawning is started. Zona radiata becomes enlarged and can be seen clearly with surrounding follicular cells. Most of the oocytes in this stage are reached to 350 μm and considerable number of oocytes reached 400-450 μm (Fig. 6b).
| Fig. 7(a-b): | Stage III: spawning stage ovary view; (a) Whole view and (b) A portion of hydrated oocyte, Yp: Yolk plate, Mn: Migratory nucleus, Zr: Zona radiata, F: Follicular cells, Total length of fish is 80 cm |
| Fig. 8(a-b): | (a) Spawning stage with different stages of oocytes and (b) Spent stage ovary Degenerating post ovulatory follicles, POF: Post ovulatory follicles, CNS: Chromatin nucleolus stage |
Stage VI: Spawning ovary
Macroscopic features: Ovaries are very large and swollen. The translucent
hydrated oocytes give the ovaries a distinctive speckled or granular appearance
through the thin ovarian wall. Eggs may be released from the ovaries when pressure
applied (Fig. 7a).
Microscopic features: Yolk granules become fused together and form yolk plates; nucleus migrates towards the periphery of the oocytes. Hydrated oocytes can be seen in the lumen. Yellow brown bodies and vascular tissue will become more prominent at this time and Post Ovulatory Follicle (POF) may be present if the fish has previously spawned. At the time of spawning ovulated eggs are found in the lumen and new POF are present in the periphery of the lamellae and few MNS, CNS, PNS may also present (Fig. 8a). Most of the oocytes in this stage are reached to 350 μm and considerable number of oocytes reached 400-450 μm (Fig. 7b).
| Fig. 9(a-b): | Atretic oocytes histology (a) α and β and (b) γ and δ stage atresia |
Stage VII: Spent ovary
Macroscopic features: Flaccid and large ovaries usually grayish in colour.
Lumen is very large.
Microscopic features: Post ovulatory follicles dominate the space; few CNS also present (Fig. 8b).
Atresia stages of oocytes were recorded in matured, Pre spawning, spawning and spent stage ovaries (Fig. 9a, b).
Testicular developmental stages
Stage I: Immature testis
Macroscopic features: Small, strap/thread like opaque testis with smooth
appearance (Fig. 10). No milt is present in the transverse
section (Fig. 10a).
Microscopic features: Testis contains spermatogonia and isolated pockets of spermatocrypts. These mainly contain spermatocytes. The central sperm sinus is small and empty (Fig. 10e).
Stage II: Maturing testis
Macroscopic features: Larger than immature gonads and produce milt when
squeezed (Fig. 10b).
Microscopic features: Spermatocytes are the prominent sperm tissues and spermatocrypts are larger than immature testis. Interstitial cells surround the spermatocrypts. Central sperm sinus is empty (Fig. 10f).
Stage III: Mature testis
Macroscopic features: Large, opaque and ivory or bone colour testis. Exterior
dorsal blood vessels are present and prominent. Produce white milt when squeezed
and milt should be visible in the outer areas of the transverse section (Fig.
10c).
Microscopic features: Spermatozoa and/or spermatids are the dominant stages. The central sperm sinus may be small with a thick muscular wall and may contain little or no sperm. However, the peripheral sperm sinuses are well developed, prominent and filled with spermatozoa (Fig. 10g, i).
| Fig. 10(a-j): | Macroscopic and microscopic staging of testes; (a) Whole view of immature testis, (b) Maturing testis, (c) Mature testis, (d) Spawning testis and histology of; (e) Spermatocytes (Scy) in spermatocrypts (Sc) of testis; (f) Large spermatocrypts, (g) Spermatocytes in peripheral sperm sinus (Pss), (h) Spermatozoa (Sz) in Pss, (i) Empty central sperm sinus (Css) and spermatozoa present in Pss and (j) Spermatozoa present in the Css |
Stage IV: Spawning testis
Macroscopic features: Running ripe stage. Testis is large in size similar
to mature stage but more have swollen with lager exterior blood vessels. Milt
is released with little or no pressure on the abdomen or when the testis is
cut (Fig. 10d).
Microscopic features: Testis is dominated by spermatozoa in the large peripheral and central sperm sinuses. Crypts of spermatocytes are uncommon and in some testis they confined to the most outer region of each lobe (Fig. 10h, j).
For oocyte diameter distribution studies, ovarian developing stages V and VI are combined as spawning stage. Frequency distribution of various size oocytes in relation to maturity stages were shown in Fig. 11. It explained that the ovaries of Scomberoides lysan have several batches of oocytes at a time but their abundance varies with months.
DISCUSSION
Macroscopic and microscopic structural changes on the ovaries of teleost were done by various researches (Saeed et al., 2010). In the present study, seven maturity stages of ovary were identified according to the macroscopic and microscopic analysis. Although, Yamamoto and Yamazaki (1961) recorded ten developmental stages of ovary and Chen et al. (2006) characterizes Thunnus orientalis oocyte development into seven stages of maturation.
Macroscopic studies based on the colour and appearance is a cheaper and faster method (Mackie and Lewis, 2001) however, microscopic histological studies on the S. lysan explain the detailed developmental changes in the gonads during maturation.
| Fig. 11(a-e): | Percentage frequency distribution of oocyte diameter at (a) Immature, (b) Maturing, (c) Rest/developing, (d) Matured and (e) Spawning stage of S. lysan, n = 6 |
In the S. lysan ovary, rest/developing and pre spawning stages were clearly identified by microscopic staging only. Proliferation of follicular cells in the peri nuclear stage of ovary explained the commencement of reproductive activity.
The presence of spawning and spent stage ovaries with hydrated oocytes and post ovulatory follicles clearly explain the spawning season as June and September. Hunter and Macewicz (1980) suggested that the best indicator of the time of spawning was the occurrence of both hydrated eggs and post ovulatory follicles. The hydration stage is very short in duration and may not be commonly observed (Stahl and Kruse, 2008; Hunter and Goldberg, 1980).
Occurrence of several stages present in an ovary at a time (Fig. 4b, 8a) revealed that the S. lysan spawns more than once. It is also supported by the studies on narrow-barred Spanish mackerel Scomberomorus commerson which has a prolonged spawning season during which eggs are spawned by females in multiple batches (McPherson, 1993) and oocytes of varying developmental stages are present within the ovary at the same time. Collection of rest/developing stage is a strong evident for the spawning pattern. That means S. lysan spawns more than once. Presence of spermatocytes in spermatocrypts of peripheral regions of spawning stage testis also explained that this species spawns more than once.
Presence of several batches of oocytes in an ovary at a time explain that S. lysan ovulated more than once termed as group synchronous ovulation pattern and it is a multiple spawner. Massut and Nin (1997) stated that the fish Coryphaena hippurus is a multiple spawner because their ovaries consist of various size distributions of oocytes with at least two groups of oocytes in the ovaries. Mohamed (2010) also explained that the presence of oocytes at different stages of development belongs to the fish with prolonged and fractional spawning season. Griffiths et al. (2005) recorded two separate peak spawning seasons in February and November for Scomberoides commersonnianus from northern Australian waters. Sadeghi et al. (2009) studied the reproductive biology of Scomberomorus commerson in the coastal waters of Iran and reported S. commerson spawn from June to September.
CONCLUSION
From the present study it can be concluded that the S. lysan is a multiple spawner with group synchronous ovulation. The spawning time was determined as June and September by identifying the presence of pre spawning, spawning and post-ovulatory follicle stages. These findings could be used in formulating or suggesting management measures for this species.
ACKNOWLEDGMENTS
The authors are grateful to the authorities of National Research Council, Sri Lanka for the financial assistance in the form of research grant (Grant No. 07-19).