HISTOMORPHOLOGICAL CHANGES IN THE OVARIES OF OREOCHROMIS NILOTICUS DURING BREEDING AND NON BREEDING SEASONS

Dept. of Anatomy and Histology,

Faculty of Veterinary Medecine, Assiut University.

Histomorphological Changes in the Ovaries Of Oreochromis niloticus

During Breeding And Non Breeding Seasons

(With 4 Tables, One Histogram and 13 Figures)

By

Enas A.A. El HafezD; Doaa M.M. Mahmoud; Sh.M. Ahmed* and A.H.S. Hassan

*Dept of Fish Diseases, Fac. of Vet. Med., Assiut University.

(Received at 19/3/2009)

التغيرات الهستومورفولوجية لمبايض البلطي النيلي أثناء موسم التکاثر

وموسم عدم التکاثر

إيناس أحمد عبد الحافظ ، دعاء محمدمختار محمود ،

شعبان محمد احمد ، احمد حسن سيد حسن

أجريت هذه الدراسة على ٥٨ عينة من إناث البلطي النيلي, وزنت هذه الأسماک بالجرام وقيست الأطوال بالملليميتر. وأعدت مبايض الأسماک للدراسة الهستولوجية بالإضافة لبعض القياسات. يتضح من الدراسة الممورفولوجية أن الأسماک لم تظهر إختلافات في الشکل الظاهري أثناء موسم التکاثر (من ابريل إلى سبتمبر) وموسم عدم التکاثر (من أکتوبر إلى مارس). وقد وجد أن لهذه السمکة مبيضان طوليان, أسطوانيان في نفس الحجم تقريبا، يقعان في الجزء الخلفي من التجويف البطنى أسفل المثانة الهوائية ويرتبطان بجدار الجسم العلوي  بمساريقا المبيض. أثناء موسم عدم التکاثر, ظهرت المبايض صغيرة بلون أصفر محمر وشغلت جزء ضئيل من التجويف البطنى حيث کان متوسط طول المبيض ٦٣ ٫ ٣±١١ سم, وقطره ٣١ ٫ ±٦1 ٫ ٣ مم, ومتوسط وزنه ٨٫٢٨±٫٥١ جم ولم نستطع رؤية البويضات. أثناء موسم التکاثر ظهرت المبايض طويلة وعريضة بلون أصفر ومليئة بالأوعية الدموية وشغلت أغلب التجويف البطنى. کان متوسط طول المبيض٠١ ٤٫ ±٢ ٫ سم, وقطره ٨٫٢±٣٦ ٫ مم, ومتوسط وزنه ٥٨ ٫ ١٢±٫٦ جم وشوهدت أعداد کبيرة من البويضات المستديرة بلون برتقالي لامع. تم تصنيف نمو البويضات إلى ستة مراحل هم: أمهات البيض، مرحلة النويات الکروماتينية، مرحلة النويات المحيطية، مرحلة حويصلات المح، مرحلة کريات المح ومرحلة النضج. ظهرت نواة المح في مرحلة النويات المحيطية، بينما ظهرت حويصلات المح محيطيا في مرحلة حويصلات المح. تميزت البويضات الناضجة بقطرها الکبير وتراکم حبيبات المح لتکوين کريات مح کبيرة ووجود حويصلات المح محيطيا في السيتوبلازم. هاجرت النواة إلى قطب الخلية ثم تکسرت عند نهاية تکوين المح. تکون جدار البويضة الناضجة من طبقة شعاعيه سميکة محاطة بالخلايا المحببة وطبقة الخلايا الغمدية. الأخيرة انقسمت إلى طبقة خارجية وداخلية. وتکرر ظهور الحويصلات المرتوقة بعد نهاية موسم التکاثر. لوحظ من خلال الدراسة النسيجية, أن مبيض البلطي النيلي يحاط بغشاء بريتونى يليه إلى الداخل المحفظة التي کانت سميکة أثناء موسم عدم التکاثر والتي أصبحت رفيعة ومليئة بالأوعية الدموية أثناء موسم التکاثر. أثناء موسم التکاثر، وجد المبيض في مرحلة تکوين المح وزاد کلا من عدد وقطر البويضات الناضجة. بينما في موسم عدم  التکاثر، أمتلئت الصفائح المبيضية بالبويضات ما قبل تکوين المح في مرحلة النويات المحيطية وايضا أمهات البيض ومرحلة النويات الکروماتينية.

SUMMARY

The present study was conducted to highlight the relationship between the external morphology of female O.niloticus and the histomorphological changes of the ovaries during different seasons of the year. A total of 58 female fishes of O.niloticus were used in this investigation. The external features of females didn't show any seasonal variations. Ovaries were paired elongated, cylindrical structure of approximate equal size, located in the posterior body cavity, ventral to the swim bladder and attached to the dorsal body wall by mesovarium. During the non-breeding season, ovaries were small, yellowish red in colour and occupied small portion of the body cavity. While during the breeding season, ovaries were extremely long and wide, yellowish in colour and more vascularized. They occupied almost the entire body cavity. The ovary of O. niloticuis was covered by a thick capsule during the non-breeding season, but became thin and vascular during the breeding period. Six arbitrary stages of oogenesis process had been establishedamong the ovarian follicles; oogonia (stagel), chromatine nucleolus stage (stage 2), perinucleolar stage (stage 3), yolk vesicle stage (stage 4), yolk globule stage (stage 5) and mature stage (stage 6). During the non-breeding season, the ovaries were filled with previtellogenic oocytes in perinucleolus stage. Oocytes in the oogonium and chromatin nucleolus stages were abundant; the oogonia reached 10+1.5/ UA and the chromatin nucleolus reached 9.8+1.4 / UA during the non-breeding season. While during the breeding season, the ovaries were in a condition of active vitellogenesis and mature oocytes increased both in number (4.0+0.5 / UA) and in diameter (806.0+11.0 µm). Morphometric studies revealed significant differences in the length, diameter and weight of the ovaries of O.niloticus during breeding and non-breeding seasons.

Key words:

INTRODUCTION

Nile tilapia belongs to genus Oreochromis. This species is naturally distributed in Palestine, the Nile River as well as most parts of African Rivers & lakes (Trewawas, 1982 and Beamish; Booth and Deacon, 2005). O. niloticus is gonochoristic, which each individual possessing a single sexual phenotype. Nile tilapia is characterized by extended spawning seasons, maturity at small size and a fast growth rate. It has been termed the aquatic chicken for its extraordinary production capabilities(Peterson; Slack; Brown- Peterson and McDonald, 2004). Tilapia have one pair of bilateral gonads locating in the posterior part of the body cavity immediately ventral to the swim bladder and attached by mesentries to the parietal peritoneum. Short ducts extend from the posterior end of the gonads to the genital pore. In addition to production of gametes, the gonads also produce hormones from endocrine tissue (Bond, 1979). The aim of the present investigation is to highlight the relationship between the external morphology of the fish and the histomorphological changes of the gonadal tissue during different seasons of the year. Also, to detect the relationship between some environmental conditions such as water temperature and spawning   activity.

Materials and Methods

The materials employed in this study consisted of randomly obtained 58 female specimens of femle Oreochromis niloticus. The materials were collected every month from the Nile River at Elkhazan bridge in Assuit city during the year. The specimens ranging from 14.46 ± 0.22 &13.92 ± 0.16 cm in standard length and from 96.39 ± 2.19 & 95.52 ± 2.00 g. in body weight. A regular record of water temperature for every month was recorded for three times using water thermometer and the mean values were taken in order to study the possible correlation between the temperature and the spawning activity of O.  niloticus(Table 1).

Ovarian measurements: The ovarian length (cm) was measured individually(right&left) usingaruler from the anterior to the posterior end. In addition the ovariandiameter (mm) was measured individually (right&left) using a caliber. Ovaries (right&left) were weighed individuallyusing Berekel balance.

Gonadosomatic index (GSI):Monthly variation of gonadosomatic index provides good indication of the extent of development of gonad with respect to the time of year (Hatikakoty and Biswas, 2004)(Table 2).

GSI was calculated monthly from each fish using the following formula:

            GSI % = Gonads (ovaries) weight (g.) / Body weight (g.) x 100          

Histological preparations:Samples for histological examination were dissected as soon as possible from the anterior, middle and posterior parts of each ovary (1x1x.05 cm) and were immediately fixed in Bouin's fluid for 24 hours. The fixed materials were dehydrated in an ascending series of ethanol, cleared in methyl benzoate and then embedded in paraffin wax.Transverse and longitudinal paraffin sections at 5-8 µm in thickness were cut and stained with the following histological stains; Harris haematoxylin and Eosin (Harri's, 1900), Grossmon's Trichrome (Grossmon, 1937), Periodic Acid –Schiff (PAS) (McManus, 1946).

Morphometrical measurements: were applied including thickness of the tunica albuginea., oocytes diameter and number/ unit area and diameter of mature ovarian follicles. By using Image analysis system (Leica Q500 MC).

Results

Morphological study:

            During the present study, the mature fish exhibited silver colouration and there was no variation in the external appearance of the fish during the breeding (from April to September) and the non-breeding seasons (from October to March). The ovaries of female O. niloticus were paired elongated, cylindrical structure and approximatly of equal size. They were located in the posterior part of the body cavity, ventral to the swim bladder. During the breeding season, ovaries were extremely long, wide and occupied almost entire of the body cavity. Large numbers of spherical bright orange oocytes were easily visible, which gave it granular texture. In addition, the ovaries were yellowish in colour and vascularized. Ripped oocytes were visible through thin transparent ovarian wall (Fig. 1). During the non breeding season, ovaries were small, ribbon- like, yellowish red in colour, occupied less than one third of the body cavity and the ova could not be seen (Fig. 2). From the present investigation, it was observed that the ovaries showed great variation in oocytes during different months of the year (Table 3)and (Histogram 1).

            There was non-significant increase in mean total and standard length of   female O. niloticus during breeding and non-breeding seasons, while there was highly significant increase in mean body weight of female O. niloticus during breeding and non-breeding seasons.

Histological study:

            The histological investigation of the ovaries of O. niloticus showed that the ovary was covered with the peritoneal membrane, which overlied the tunica albuginea that consisted of vascular collagenous connective tissue (Fig. 3) elastic fibers and smooth muscle fibers. The ovarian cavity (ovocoel) remained in the center of the ovary as irregular space lined with germinal epithelium (simple squamous epithelium), which divided to give oogonia. The ovocoel continued posteriorly as the lumen of the oviduct (Fig. 4 a,b). The developing ova were held together by the stroma (vascular collagenous connective tissue and few strands of smooth muscle fibers). The stroma consisted of finger- like ovarian (ovigerous) lamellae which contained ovarian follicles at different stages of oogenesis in addition to atretic follicles (Figs. 4 a&b). Ovigerous lamellae protruded into the ovocoel from the ovarian wall and oogenesis occurred in these lamellae.

a)- Oogenesis: Oogenesis involved the proliferation of oogonia by mitosis and the development of oocytes. According to changes in size, nucleus, ooplasm and egg membranes of the developing ova, six stages were observed in           O. niloticus: Stage 1- Oogonia:They were found in groups or nests in the ovigerous lamellae, associated with the germinal epithelium and they were the smallest cells of the germinative lineage. They were small spherical cells with large light basophilic nucleus with a single nucleolus. Thin film of faintly stained ooplasm surrounds the nucleus (Fig. 5a&b). All ovaries of developing and mature females had several patches of oogonia Oogonia divided by mitosis to give primary oocytes.

            All ovaries of developing and mature females had several patches of oogonia. Oogonia divided by mitosis to give primary oocytes. During the breeding seasons, they reached 6.4 ± 2.1 in number / unit area and 28 ± 0.8 µm in diameter while during the non-breeding seasons, they reached 10 ± 1.5 in number / unit area and 24.8  ±2.0 µm in diameter (Table 3).Stage 2- Early oocyte (chromatin nucleolus stage): Transformation of oogonia to oocyte includes increase in the size of the cell and nucleus (Table 3).Chromatin of the nucleus appeared thread like and distributed throughout the nucleus. The number of nucleoli increased which distributed throughout the nucleoplasm. The ooplasm was reduced and deeply basophilic. These oocytes had little or no affinity with dyes used (Fig. 6). During the breeding seasons, they reached      3.2 ± 0.50 in number / unit area and 44.0 ± 2.4 µm in diameter while during the non-breeding seasons; they reached 9.8 ± 1.4 in number / unit area and         40.0 ± 2.4 µm in diameter (Table 3). Early oocytes showed highly significant decrease in number / unit area but non-significant increase were found in diameter in the breeding seasons. Stage 3- Late oocyte (perinucleolar stage):                          The nucleus stained lightly and the number of nucleoli increased, arranged themselvs in the peripheral part of the nucleus. Chromosomes were assembled at one side of the nucleus. Ooplasm contained yolk nucleus (Balbiani bodies), which appeared as basophilic round mass. Oocyte surrounded by a single layer of squamous follicular epithelium (Figs.5c&7). Oocyte increased progressively in number and diameter (Table 3). During the breeding seasons, they reached 4.0 ± 1.1 in number / unit area and 100.0 ± 6.1 µm in diameter, while during the non-breeding season, they reached 9.0 ± 2.4 in number / unit area and 98.0 ± 3.8 µm in diameter. Late oocytes showed significant decrease in number but non-significant increase were found in diameter in the breeding season (Table 3).Stage 4- Vacuolated follicles     (yolk vesicle or cortical alveolar stage): Nucleus increased in size to become the germinal vesicle. Nucleoplasm attained acidophilic reaction and the nuclear membrane became irregular.Ooplasm became faintly basophilic and yolk vesicles were peripherally arranged (PAS positive), late become cortical alveoli and take part in the formation of perivitelline space. Yolk granules appeared in the ooplasm around the nucleus. Zona radiata (oolemma) begin to appear which was acellular thin hyaline acidophilic membrane. The follicular layer was formed of cuboidal cells. The stroma of the ovary was formed of flat thecal cells (thin layer of fibroblasts) that surrounded the follicular layer (Figs. 8). Oocytes increased both in number and diameter (Table 3). During the breeding season, they reached 5.4 ± 2.2 in number / unit area and 242.0 ± 7.0 µm in diameter while during the non-breeding season, they reached 5.0 ± 0.4 in number / unit area and 204.0 ± 7.3 µm in diameter. Late oocytes showed non-significant decrease in number but highly significant increase were found in diameter in the breeding season (Table 3). Stage 5- Yolk globule stage (vitellogenesis): Yolk vesicles increased in size. Yolk granules accumulated into large yolk globules. Several rounded yolk globules (platelets) appeared near the center of the oocyte and extend centrifugally until only a thin peripheral shell of cytoplasm remains. These globules gave PAS positive reaction. Zona radiata appeared thick and the follicular epithelium (granulosa) made up of cuboidal cells. Theca folliculi divided into outer vascular, collagenous connective tissue thecal layer and inner cellular theca cells       (Fig. 9). Basal lamina (thin fibrous layer, PAS positive) was found between follicular epithelium and theca layer. When vitellogenesis begins, the egg   membranes became clear and PAS positive. Oocyte increased in size (Table 3). During the breeding seasons, they reached 4.0 ± 0.31 in number / unit area and 492.0 ± 2.7 µm in diameter while during the non-breeding season they reached 2.0 ± 0.6 in number / unit area and 313.0 ± 6.0 µm in diameter. Yolk globule oocytes showed highly significant increase in number and significant increase were found in diameter in the breeding season.Stage 6- Mature follicles:Many empty large vacuoles found towards oocyte periphery and the yolk globules increase in size. The nucleus gradually disappeared and begin to migrate to the animal pole (germinal vesicle migration stage) which usually occur at the end of vitellogenesis (Figs. 11 a,b,c,d). At the end of this stage, Zona radiata attained its maximal thickness surrounded by granulosa and theca cells.Strong PAS positive reaction was observed in the cytoplasm of mature follicles between yolk globules (Figs. 10 a,b,c &11 d). Oocyte was large in diameter, they reached 1049.5 µmin May (Table 3). During the breeding season, they reached 4.0 ± 0.5 in number / unit area and its mean 806.0 ± 15.0 µm in diameter while during the non-breeding season, they reached 1.2 ± 0.2 in number / unit area and 509.0 ± 4.0 µm in diameter. Mature oocytes showed significant increase in number and highly significant increase were found in diameter in the breeding season (Table 3).

b) - Atretic follicles: Its diameter was too variable to be measured, since it was irregular structure that could be seen at any time during oocyte maturation, but it always formed at the period of post-spawning in oocytes at any stage.These follicles characterized by breakdown of oolemma, yolk liquefaction, presence of many empty spaces, the granulosa hypertrophied and their cells increased in number and the connective tissue surrounding the follicles thickened and become more vascular and appearance of small acidophilic mass of unclear structure in the cytoplasm of these follicles to give structure resemble corpora atretica or yellow brown bodies (Fig. 12). First oocytes to be reabsorbed were those containing yolk.

Histological variation of the ovarian tissue during the breeding and non- breeding seasons:

a) - During the non-breeding seasons:

            The tunica albuginea was thick during the non- breeding seasons and contained much amount of smooth muscle fibers (Fig. 13), its mean thickness reached 48.01 ± 4.56 µm(Table 4) and the maximum mean value was 72.18 ± 0.79 µm in February.

            The ovarian lamellae were thin in non-breeding seasons. The lamellae were filled with previtellogenic oocytes in perinucleolus stage. Oocytes in the oogonium and chromatin nucleolus stage were abundant, while mature ovarian follicles were absent in January (Table 3). The lamellae were disrupted and disorganized with several empty spaces and extensive vascularization. Remnants of atretic follicles were present throughout the ovary (Fig.12).

B)- During the breeding seasons:

            The tunica albuginea was thin and vascular, its mean thickness reached 29.18 ± 0.67 µm(Table 6).

            The ovarian lamellae were thick and completely obliterated ovaries. Mature ovary was in active vitellogenesis. Oocytes in all stages of development, from perinucleolus to ripe stages could be identified, but late stages of vitellogenesis were dominant (Figs. 4 a&b).

            Mature oocytes increased both in number to reach about 5 / unit area in May and July and also in diameter to reach about 1049.5 µm in May, while perinucleolar oocytes were absent in June and July (Table 3). Stroma of mature breeding ovary was pressed due to enlargement of ocytes (Figs. 4 a).

Gonadosomatic index:

   GSI= gonads weight \ body weight x 100

            GSI can be used as indicator for gonadal development. When gonadosomatic index reach a maximum value, this gives a perfect indication to the time of spawning.

Gonadosomatic indix were highest between   May to July. GSI peaked in June   (Table 1).   

            In our study we found that the mean GSI during the breeding seasons   was 12.58 ± 0.59 %. While the mean GSI during the non- breeding seasons was 9.17 ± 0.36 % (Table 1). From the present investigation, the water temperatures were highly correlated with GSI and the optimum temperature for spawning was between 21 ^oC to 24 ^oC (from May to July) (Table 2) that was the peak period for the GSI.

Table 1: Average monthly fluctuations in GSI.

Table 2: The mean values of water temperature during different months of the year.                                               

Table 3: Number and diameter (µm) of all stages of oocyte development during different months of the year / unit area.

n.s means the values are non- significant.

* means the values are significant. ** means the values are highly significant.