Advances in Environmental Biology

Advances in Environmental Biology, 9(8) May 2015, Pages: 210-215

AENSI Journals

Advances in Environmental Biology ISSN-1995-0756

EISSN-1998-1066

Journal home page: http://www.aensiweb.com/AEB/

Morpho-histology of the Reproductive Duct in Short Mackerel Rastrelliger brachysoma (Bleeker, 1865) 1Sinlapachai 1 2

Senarat, 1Jes Kettratad, 2Wannee Jiraungkoorskul

Department of Marine Science, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand Department of Pathobiology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand

ARTICLE INFO Article history: Received 12 December 2014 Received in revised form 26 January 2015 Accepted 23 February 2015 Available online 28 May 2015 Keywords: Fish, Histology, Mackerel, Oviduct, Rastrelliger brachysoma, Thailand

ABSTRACT Background: Short mackerel, Rastrelliger brachysoma (Family Scrombidae), is one of the most important marine fish and a new candidate for aquaculture in Thailand. It requires the knowledge especially reproductive duct. Hence, basic histological and histochemical properties of this structure were investigated in the sexually matured. Fishes were taken during October to December 2013 from the Upper Gulf of Thailand. The histological results revealed that the oviductal morphology consisted of a tubular organ, which was joined with the ovaries in the dorsal part. It was composed of tunica mucosa, tunica submucosa, tunica muscularis and tunica serosa layers. The epithelial layer of the mucosa was lined by low simple columnar epithelium and contained different cell types (ciliated epithelial, mucous secreting and basal cells). Among the mucosal layer, the secretory cell was positively stained with periodic acid-schiff and aniline blue, which indicated that the secretory cell produced the glycoprotein and the mucopolysaccharide. Other characterizations of the three layers were also described and discussed in our study. The testicular duct in mackerel male was observed in similar to oviduct under light microscopic level. Conclusion: Our finding concluded that four distinct layers were histologically observed in the reproductive duct of R. brachysoma, which is novel and will be useful to support in further studies.

© 2015 AENSI Publisher All rights reserved. To Cite This Article: Senarat, S., Kettratad, J., Jiraungkooskul, W., Morpho-histology of the reproductive duct in short mackerel Rastrelliger brachysoma (Bleeker, 1865). Adv. Environ. Biol., 9(8), 210-215, 2015

INTRODUCTION Several investigations concerning the reproductive duct in teleost fish have reported with composing of oviductal female and testicular duct in male fish [6]. A details assessment of the teleostean oviduct is considered a part of the reproductive system, which plays an important role in assisting gamete transportation (egg and sperm) [14]. Previous observations on the oviduct structure have been made in several fish species, including Alburnus alburnus [12], Rhinobatos lentiginosus [10] and Xiphophorus maculatus [15]. These studies have provided vital information about histological structure under light microscopic level. The oviductal wall is principally composed of four layers; mucosa composing two sub-layers (epithelium and lamina propria), submucosa, muscular and serosa layer (or peritoneum). Studies show the several cell types are lined by ciliated and non-ciliated epithelial and secretory cells. It possible that only secretory cells produce the fluid that plays an important role during transport of gametes [14]. As mention above, many studies about the reproductive duct have been especially conducted on the oviductal structure, which is of great interest to several investigators with using to apply in aquaculture [10, 15]. On the other hand, the testicular duct is rarely structural reported in number of fish species such as Cichla Kelberi [17] and Acanthopagrus schlegeli [13], which needs to be clarified. Rastrilliger brachysoma is considered a very economically important marine fish and a new candidate for aquaculture in Thailand. It is necessary to define its reproductive biology and the reproductive system. Unfortunately, the reproductive structure of this species has never been reported, in spite of investigation of gonadal structure and gametogenesis (Senarat et al., in progress). In the present study, the reproductive duct of R. brachysoma from the Upper Gulf of Thailand was continuously described to gross anatomy and structural details, as revealed by histological and histochemical techniques. A comprehensive investigation of these Corresponding Author: Assistant Professor Dr. Wannee Jiraungkoorskul, Department of Pathobiology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand. Tel: (+66) 02-201-5550; Fax: (+66) 02-354-7158 E-mail: [email protected]

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Wannee Jiraungkoorskul et al, 2015 Advances in Environmental Biology, 9(8) May 2015, Pages: 210-215

regions could be useful to support other studies, such as fine structure, immunochemical activities and comparative phylogenetic studies in the scrombridae group.

Fig. 1: (A) Map of the Upper Gulf of Thailand, Samut Songkhram Province, Thailand showing our study area. MATERIALS AND METHODS Animal collection: Ten sexually mature, R. brachysoma with standard length of approx. 16.5-20.0 cm were collected in the fishing season (October to December 2013). All fish were captured by bamboo strake trap from Samut Songkram Province (13°16’18.4” N, 100°02’13.4” E) (Figure 1). The identification and collection were performed based on the identification key of FAO [8]. The experimental protocol was approved by the Animal Care and Use Committee of the Faculty of Science in accordance with the guide for the care and use of laboratory animals prepared by Chulalongkorn University (Protocol Review No. 1423003). Tissue preparation of histological examinations: These fish were euthanized by rapidly cooling shock [19] and then gonadal tissues were carefully kept and separated for fixation in Davidson’s fixative solution. Anatomical nomenclature of the gonadal tissue was evaluated under stereo microscope. All samples were performed at the Laboratory of Fish Research Unit, Department of Pathobiology, Faculty of Science, Mahidol University, Thailand. The sections were cut at 6-7 µm thickness. Histological sections were histologically stained with Delafield's hematoxylin and eosin (H&E) as well as histochemically stained with Masson’s trichrome (MT) to identify the muscle and collagen fibers, periodic acid-schiff (PAS) and aniline blue (AB) pH 2.5 to identify the basic structure and chemical compositions [3, 11]. Another investigation of the semithin section, small pieces of oviduct were suddenly dissected, rapidly prefixed in 2.5% glutaraldehyde and postfixed in 1% osmium tetroxide. All specimens were dehydrated and embedded in Epon 812 epoxy resin. Semithin sections were cut at 500 nm thicknesses and stained with 0.5% toluidine blue for orientation. Under histological sections and semithin sections, a reproductive structure was examined under light microscope (Canon EOS 1100D digital camera). RESULTS AND DISCUSSION Morphology and histology of oviduct: The gross anatomy of the oviduct of R. brachysoma had two lateral parts: left and right oviducts. Each oviduct, as a thin and tubular organ was connected with the ovary in the dorsal part before joined into the genital duct and cloaca in the ventral part (Figure 2). This anatomical structure agreed with previous reports of Detrich and Krieger [6] and Selman and Wallace [16], who discussed the oviduct in some teleost fish. However, it could be not classified into four major regions: oviduct, nidamentary gland, uterus and vagina, which are principally found in Dasyatis bleekeri [5]. The significance of the uterus and vagina in the oviduct has not only been shown in chondrichthyes [7], but also in lizards, including Crotaphytus collaris and Eumeces obsoletus [9]. At light microscopic level in both section and semithin section, the basic histological organization was similarly shown along the oviduct. There were four layers, including tunica mucosa, tunica submucosa, tunica muscularis and

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tunica serosa layers that were shown in the oviductal wall (Figure 3A-B, 3G-H). These results were similar to those described in several fish including A. alburnus [12], R. lentiginosus [10] and X. maculatus [15].

Fig. 2: Gross and illustration morphology (a) and light micrograph (A-B) of the oviduct (Ov) of Rastrilliger brachysoma during non-breeding season. Note: Ca-cloaca, O-ovary, Mv-mesovarium. Scale bar in a=1cm, A-B=350μm. Masson’s trichrome (MT). The mucosal layer as well as the innermost layer was composed of a few longitudinal folds, referred to as fimbriae. Folds were branched to different degrees, becoming primary and secondary folds. Also, this mucosal layer was composed of two sub-layers: epithelial layer and lamina propria. High magnification of a section of the epithelial layer showed it was covered by low simple columnar epithelium. This presence of oviduct structure was similarly reported in another fish, A. alburnus [12]. Moreover, three different cell types with different heights were identified, including ciliated epithelial, mucous secretory and basal cells (Figure 3C-D). The first cell type, the most abundant of the ciliated epithelial cells, was the elongated cell containing cilia. A prominent nucleus was basophilic stain and surrounded by eosinophilic cytoplasm. However, its function and mechanism is unknown. The second cell type, the mucous secretory cell, was located among the epithelial cell, but unlike epithelial cell because it was positively detected in the AB and PAS reactions. It was indicated that the secretory cell produced mucopolysacchraide and glycoprotein. As reported in a previous study, the role of these substrates may be to produce and secrete during gamete transport [14]. Additionally, the appearance and the secretion of the glycoprotein profile are also suggested to be related to physiological function and ovarian hormones [4]. The mucous secreting cell in R. brachysoma may have a close relation to the non-ciliated cells of the oviduct, which are found in other vertebrates including Chinese Meishan pig [1], some other mammals [2] and Bos Taurus [18] because this cell synthesizes and releases the glycoprotein. Therefore, this might be an evolutionary trend that conserves the structural function of this cell in the oviductal epithelium. The final cell type, the basal cell, was mostly located between the epithelial layer and the basement membrane. It was less frequent and relatively smaller in size. Under the epithelial layer in the present study was the thin layer of lamina propria, which contained various compositions, particularly fibroblasts and loose fibrous connective tissue. This connective tissue was positively stained, as shown by MT technique. Underneath the mucosal layer, the thin layer of submucosa contained the loose connective tissue and several blood vessels. Our findings showed the tunica muscularis layer in this species was composed of two smooth muscle layers: inner circular muscle and outer few longitudinal muscles. It had clearly defined the smooth muscle fibers. It is possible that tunica muscularis layer was associated with movement of the oviduct structure. Another important finding was that the thin layer of serosa finally surrounded the tunica muscle, forming simple squamous epithelium, a small amount of loose connective tissue and nerves to gather with many blood vessels. Morphology and histology of testicular duct: In a study involving the morphological localization of the testicular duct of R. brachysoma, it descended along the lateral side of testis and then it connected with cloaca (Fig. 3A). Histologically, section of this

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structure was found similar to oviductal female, which was composed of tunica mucosa, tunica submucosa, tunica muscularis and tunica serosa, in accordance with an observation in another fish [17]. No longitudinal fold in the mucosal surface was observed in all specimens with lining by low simple columar epithelium. This layer was surrounded by tunica submucosa, which was rarely identified in testicular duct. The layer of the tunica muscularis showed within duct with consisting of a few muscular tissue, whereas the serosa was slightly seen in this duct. Through this duct, the mature sperm as well as final sperm maturation were also found (Fig. 3B-D). Conclusion: Demonstration of the reproductive structure is rarely found in the literature review of teleost fish as well as scrombidae. This study established this for the first time in R. brachysoma and revealed the histological characterization of the reproductive duct, which was composed of tunica mucosa, tunica submucosa, tunica muscularis and tunica serosa. Comparative changes of histological structure in the ducts during breeding and non-breeding seasons in R. brachysoma will be explored in further studies, including length and numbers of the cell types.

Fig. 3: Illustration morphology (A, D) and light micrograph of the oviduct (Ov) of Rastrilliger brachysoma. Note: Bc-basal cell, Bv-blood vessel, Cm-circular muscle, e-epithelial cell, Lm-longitudinal muscle, Lplamina propria, m-tunica mucosa, M-tunica muscularis, Mc-mucous secreting cells, O-ovary, S-tunica serosa, Sm-tunica submucosa. Scale bar in B, E and F=100μm, C=20μm. Masson’s trichrome (MT), Periodic Acid-Schiff (PAS) and aniline blue (AB).

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Fig. 4: Gross morphology (A) and light micrograph (B-D) of the testicular duct (Td) of Rastrilliger brachysoma. Note: Bv-blood vessel, Ca-cloaca, m-tunica mucosa, M-tunica muscularis, s-tunica serosa, Sm-tunica submucosa, Sl-seminiferous lobule, Sz-spermatozoa. Scale bar in A=0.3cm, B=200μm, C-D=50μm. ACKNOWLEDGMENTS This work was supported by the 100th Anniversary Chulalongkorn University Fund for Doctoral Scholarship. The authors express gratefulness to Ms. Piyakorn Boonyoung, Dr. Niwat Kangwanrangsan and Dr. Watiporn Yenchum for suggestions and comments about histochemical techniques. We gratefully acknowledge the members of the Fish Research Unit, Department of Pathobiology, Faculty of Science, Mahidol University. Also, we thank Mr. Mark Tunmore for critically reading the manuscript. REFERENCES [1] [2] [3] [4] [5]

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