Int. J. Aquat. Biol. (2016) 4(1): 31-42 E-ISSN: 2322-5270; P-ISSN: 2383-0956 Journal homepage: www.ij-aquaticbiology.com © 2016 Iranian Society of Ichthyology Original Article Morpho-histological characteristics of gonads and reproductive index in an endemic fish species, Oxynoemacheilus persa (Heckel, 1847) (Teleostei: Nemacheilidae) from Kor River basin, Iran Soroor Mirghiyasi, Hamid Reza Esmaeili*,1Mohsen Nokhbatolfoghahai Ichthyology and Developmental Systematics Lab., Department of Biology, College of Sciences, Shiraz University, Shiraz, Iran. Article history: Received 8 November 2015 Accepted 19 January 2016 Available online 2 5 February 2016 Keywords: Reproduction Gonad morphology Gonad histology Sexual maturation Gonado-somatic index Abstract: This study presents the first details on morphological and histological characteristics of gonads, gonadal development stages and reproductive index of the Persian loach, Oxynoemacheilus persa (Heckel, 1847), an Iranian endemic species. Sampling was done from April 2010 to April 2011 using electrofishing device and a total of 196 individuals were collected. The gonads of specimens were removed, their sexes determined and then were fixed in 10% formalin solution after checking their morphology and measuring their weights, lengths and widths. Based on the size, shape and weight of the gonads, degree of occupation of the body cavity, presence or absence of ripe oocytes or milt, diameter of the oocytes in the ovary, and histological observations, five stages of sexual maturation in females and four stages in males were determined by macroscopic and microscopic criteria. The results of the gonadal stages showed that the O. persa spawns in the middle of spring and these stages were correlated to the gonado-somatic index (GSI). Introduction The order Cypriniformes with 11 family and 4298 species is one of the large order of fishes (Eschmeyer and Fong, 2016). The family Nemacheilidae comprises about 659 species which is the second largest family in the order Cypriniformes (Eschmeyer and Fong, 2016). The nemacheilid fishes are found throughout Eurasia and northeast Africa (Berra, 2001; Nelson, 2006). They are small fishes, live in fast flow hill streams with quite secretive life hiding under stones or in mud. The genus Oxynoemacheilus with about 47 species, is a species-rich genus of nemacheilids known from Albania eastwards to Central Iran (Golzarianpour et al., 2011; Erk'akan, 2012; Kottelat, 2012; Kamangar et al., 2014; Mafakheri et al., 2015). The Persian loach, Oxynoemacheilus persa (Heckel, 1847) which is generally known as Persian sag mahi (= dog fish), louch (= louch) or mar mahi parsi (= snake fish) in Farsi, is an endemic poorly known loach distributed in endorheic Kor and * Corresponding author: Hamid Reza Esmaeili DOI: http://dx.doi.org/10.7508/ijab.2016.01.005 E-mail address: hresmaeili@shirazu.ac.ir exorheic Persis basins in southern Iran (Esmaeili et al., 2010, 2015) and information about different aspects of its biology and ecology including its reproduction is scare. Reproductive studies of fishes, such as assessment of size at maturity, duration of the spawning season and fecundity, require knowledge of the state of gonad development and a large number of macroscopic maturity scales in individual fish (Carrasson and Bau, 2003). Although macroscopic staging can enable detailed recording of the seasonal occurrence of different reproductive stages, histological analysis of the gonads can provide a more precise determination. Cellular substructures can be recognized in the growing follicles and ovarian tissue and allow for unambiguous grading and interpretation of reproductive status. Description of the general pattern of histology and development of teleost are given by Wallace and Selman (1981), Tylerand Sumpter (1996), and Tomkiewicz et al. (2003). Since 32 Mirghiyasi et al./ Morpho-histological characteristics of gonads in Oxynoemacheilus persa there is no data available regarding the gonadal morphology and histology of Oxynoemacheilus persa, therefore, this study was conducted to presents a detailed description of gonad morpho-histology of this species. Materials and Methods A total of 196 specimens of O. persa (Fig. 1) (81 males and 111 females) were collected monthly from Kor River basin, Fars Province (30°36'16.9'' N; 52°56'40.1'' E) from April 2010 to April 2011 using electrofishing device. After anesthesia, fish were fixed into 10% formalin in the field, then labeled individually and deposited in the Zoological Museum, Collection of Biology Department, Shiraz University (ZM-CBSU). Some morphological measurements were made using a digital caliper to the nearest 0.01 mm and body weights were determined by a digital balance to the nearest 0.001 g. For histological studies, the specimens were dissected and their ovary or testis were removed. The sexes and stages of sexual maturation were determined as possible as by naked eye examination and under a compound microscope (Olympus). The chi-square test was used to assess deviation from 50:50 sex ratio based on Robards et al. (1999). Weight, length, width, color, and shape of each gonad were recorded and the maturity stage of them was recognized macroscopically based on Nikolsky (1963). The histological sections of ovary or testis of each maturing stages were prepared by routine histology method (Bancroft and Stevens, 1991; Eagderi et al., 2013) as follow: they dehydrated in alcohol, cleared in xylene, imbedded in paraffin wax at 56°C melting point, sectioned at 5-7 μm thickness, and then the sections were stained by Hemotoxylin and Eosin (H&E) staining method. The histological slides were studied under a light compound microscopy and their pictures were taken by a compound microscope equipped to a digital camera. The gonado-somatic index (GSI) was calculated by dividing the gonads weight by the whole body weight and multiply by 100 (Nikolsky, 1963) to examine the seasonal changes in the gonads for estimating spawning season. Results Size range: A total of 196 individuals of O. persa were collected ranging 25.19-78.24 mm (S.D: 12.48) in total length, 24.29-74.22 mm (S.D: 12.02) in fork length, 21.34-66.01 (S.D: 10.55) in standard length and 0.085 to 4.50 g (S.D: 0.90) in total weight. Sex ratio: The collected 81 male and 111 female specimens giving an overall sex ratio of 1M: 1.37F. It was significantly female biased (Chi square =12.629, P<0.001). Gonado-somatic index: The results showed that female invest more in gonads than males (ANOVA, P<0.001). A significant differences were observed between female and male’s gonado-somatic index (ANOVA, P<0.05). Both female and male gonado- somatic indices peaked in April showing that Figure 1. Oxynoemacheilus persa, from Kor River basin, Fars Province. 33 Int. J. Aquat. Biol. (2016) 4(1): 31-42 Figure 2. Monthly variation of mean GSI in female and male specimens of Oxynoemacheilus persa. 34 Mirghiyasi et al./ Morpho-histological characteristics of gonads in Oxynoemacheilus persa O. persa spawns in the middle of spring (Fig. 2). Macroscopic and microscopic characteristics of ovaries: The left and right ovaries of O. persa were stuck to each other and made an integrated ovary extending along the body cavity in dorsal position above the gut. Based on the size and weight of the ovary, degree of occupation of the body cavity, presence or absence of ripe oocytes, diameter of the oocytes in the ovary, and histological observations, we described 5 maturation stages in the ovary. The stages were classified as immature (Stage I), immature and developing (Stage II, early and late), maturing (Stage III), ripe (Stage IV), spawning (stage V). No specimens of Immature stage were observed and the stage II was divided into two stages: early and late. Gonad measurements of O. persa have shown in Tables 1 and 2. Ovary maturation stages: No specimens of Immature stage (I) were observed. In the stage II, early immature, the ovary was small and cream in color. Oocytes were not visible to the naked eye. The mean diameter of oocytes was 0.073 mm (Fig. 3a). In histological sections of the ovary, small and round oocytes with a large nucleus and abundant nucleoli around the nucleoplasm were observed. No lipid droplets were found in this stage. The oocytes had basophilic cytoplasm and an acidophilic nucleus. The ratio of nucleus to cytoplasm volume was high (Fig. 4a, b). In the developing stage (II, late), the ovary was larger and the oocytes were not visible by naked eye. The mean diameter of oocytes was 0.131 mm (Fig. 3b). In histological observations, the oocyte size increased and the ratio of nucleus to cytoplasm decreased. In larger oocytes, the lipid droplets were formed in the cytoplasm and follicular and theca layers covered each oocyte. In addition, a very thin zona radiate appeared between oolema and follicular layer. The zona radiata was distinguished in two parts: One part as finger-like projections which is placed under the follicular cells and another Stage Measurements N Minimum Maximum Mean Std. Deviation 2 early Gonad Weight (g) 19 0.002 0.013 0.00532 0.002868 Gonad length (mm) 19 5.010 9.600 6.87211 1.150447 Gonad Width (mm) 19 1.273 2.543 1.74737 0.297476 GL.ACL (%) 19 44.97 76.49 57.8247 7.54236 2 late Gonad Weight (g) 39 0.030 0.188 0.06713 0.025999 Gonad length (mm) 39 10.050 19.380 14.04615 1.932407 Gonad Width (mm) 39 2.257 5.237 3.18667 .472447 GL.ACL (%) 39 51.65 78.38 68.9231 5.69150 3 Gonad Weight (g) 14 0.017 0.202 0.10521 0.054044 Gonad length (mm) 14 10.050 20.170 15.40643 2.931255 Gonad Width (mm) 14 2.033 4.597 3.67643 .847645 GL.ACL (%) 14 48.41 96.72 74.6653 13.82134 4 Gonad Weight (g) 18 0.144 0.423 0.24367 0.073964 Gonad length (mm) 18 13.690 20.880 18.14833 1.783800 Gonad Width (mm) 18 4.493 6.093 5.14926 .426514 GL.ACL (%) 18 63.26 99.06 89.9383 9.06568 5 Gonad Weight (g) 21 0.234 0.795 0.42562 0.131349 Gonad length (mm) 21 18.140 29.190 20.60238 2.261541 Gonad Width (mm) 21 4.907 10.367 6.70921 1.133205 GL.ACL (%) 21 79.94 99.43 91.7612 5.21365 GL.ACL: The ratio of gonad length to abdominal cavity length. Table 1. Descriptive statistics of gonad measurements in Oxynoemacheilus persa (female) from Kor River basin, Fars Province. 35 Int. J. Aquat. Biol. (2016) 4(1): 31-42 Figure 3. Morphology of ovary of Oxynoemacheilus persa in different stages. (a) Immature stage (II, early), (b) Developing stage (II, late), (c) Maturation stage (III), (d) Ripe stage (IV), and (e) Spawning stage (V). Stage Measurements N Minimum Maximum Mean Std. Deviation 1 Gonad Weight (g) 24 0.001 0.002 0.00113 0.000338 Gonad length (mm) 24 4.28 11.20 6.2642 1.61155 Gonad Width (mm) 24 0.060 0.302 0.16389 0.055755 GL.ACL (%) 24 0.92 4.29 2.1816 0.77465 2 Gonad Weight (g) 19 0.001 0.021 0.01011 0.004665 Gonad length (mm) 19 3.62 11.19 7.3453 2.02354 Gonad Width (mm) 19 0.413 1.175 0.61404 0.176038 GL.ACL (%) 18 3.07 9.25 4.4624 1.51573 3 Gonad Weight (g) 18 0.010 0.028 0.01689 0.005075 Gonad length (mm) 18 6.16 11.85 8.2925 1.32283 Gonad Width (mm) 18 0.492 0.912 0.65546 0.123627 GL.ACL (%) 18 3.22 6.45 4.5645 0.89245 4 Gonad Weight (g) 19 0.016 0.070 0.03642 0.014261 Gonad length (mm) 19 7.73 16.48 11.1745 2.25921 Gonad Width (mm) 19 0.613 1.158 0.90167 0.168485 GL.ACL (%) 19 4.08 7.25 5.8294 .96958 GL.ACL: The ratio of gonad length to abdominal cavity length. Table 2. Descriptive statistics of gonad measurements in Oxynoemacheilus persa (male) from Kor River basin, Fars Province. 36 Mirghiyasi et al./ Morpho-histological characteristics of gonads in Oxynoemacheilus persa uniform thin part on the oocyte membrane. This stage is called returning stage, because after spawning, the ovaries, return to this stage to start oogenesis (Fig. 4c, d). In the third stage i.e. maturing stage, the ovary size was clearly larger than the previous stage and its color changed from cream to light yellow due to accumulation of the yolk materials in the oocytes. Yellow oocytes with an average diameter of 0.237 mm were visible to the naked eye in this stage (Fig. 3c). The growing oocytes were characterized by small acidophilic yolk granules and many clear lipid droplets that had entirely filled the cytoplasm and also thickened two parts of zona radiata. The finger-like projections were significantly elongated (Fig. 4e, f). In the ripe stage (IV), the size and weight of Figure 4. Microphotographs of ovaries of Oxynoemacheilus persa in different stages. (a-b) Immature stage (II, early), (c-d) Developing stage (II, late), (e-f) Maturation stage (III), (g-h) Ripe stage (IV), and (i-j) Spawning stage (V). N: Nucleus, Nu: Nucleoli, LD: Lipid Droplets, TL: Theca Layer, FCL: Follicular cell Layer, FLP: Finger–Like Projections, and ZR: Zona Radiata. 37 Int. J. Aquat. Biol. (2016) 4(1): 31-42 gonads increased but had not achieved their maximum size. Their color was yellow, and also they wrinkled a little. The mean of ova diameter was 0.442 mm (Fig. 3d). In this stage, the oocytes are known as follicles. Their size increased and the ratio of nucleus to cytoplasm decreased. The large follicles were full of lipid droplets more than pervious stages. Furthermore in this stage, a coalescence of lipids and yolk granules were occurred. The nuclear envelope broke down and the thickness of the zona radiata was higher than previous stages (Fig. 4g, h). In the spawning stage (V), the ovary was yellow and occupied most of the body cavity. It achieved its maximum weight. Large and yellow oocytes, full of yolk with an average diameter of 0.518 mm were distinguishable in this stage (Fig. 3e). Cohesion between oocytes decreased and some of them were observed separately in the body cavity. Oocytes were characterized by large mass of yolk and numerous large lipid droplets. The zona radiata was completely thick in this stage (Fig. 4i, j). Testes maturation stages: Based on macroscopic and microscopic observations, 4 stages of maturation were distinguished for males of O. persa, as follow: Immature stage (I): The testes were very thin, thread- like and grey in color (Fig. 5a). Spermatogonia were the dominant cells. These cells were the largest spermatogenic cells, with clear cytoplasm and large nucleus. Some primary spermatocytes were also observed (Fig. 6a). Developing stage (II): The size of testes increased (Fig. 5b). Spermatogonia, primary and secondary spermatocytes were more remarkable in the histological sections. Secondary spermatocytes were similar to the primary spermatocytes but smaller (Fig. 6b). Maturing stage (III): The testes were more elongated, flat and milky (Fig. 5c). The number of spermatogonia were significantly reduced. Primary and secondary spermatocytes, and spermatids were observed in the tubules. These cells were located in clusters. Spermatids were smaller than the secondary spermatocytes (Fig. 6c). Ripe stage (IV): Testes were quite milky and massive (Figure 5d). Tubules were characterized by Figure 4. Continued. 38 Mirghiyasi et al./ Morpho-histological characteristics of gonads in Oxynoemacheilus persa having secondary spermatocytes, large numbers of spermatids and spermatozoa. The predominant cells were spermatozoa with a dark blue stain related to their nucleus. They were the smallest spermatogenic Figure 5. Morphology of testes of Oxynoemacheilus persa in different stages. (a) Immature stage (I), (b) Developing stage (II), (c) Maturation stage (III), and (d) Ripe stage (IV). Figure 6. Microphotographs of testes of Oxynoemacheilus persa in different stages. (a) Immature stage (I), (b) Developing stage (II), (c) Maturation stage (III), and (d) Ripe stage (IV). S: Spermatogonia, PS: Primary Spermatocytes, SS: Secondary Spermatocytes, St: Spermatids, and Sz: Spermatozoa. 39 Int. J. Aquat. Biol. (2016) 4(1): 31-42 cells (Fig. 6d). Discussion This study provided the details on gonad morphology and histology of O. persa, an endemic loach species of Iran. Oxynoemacheilus persa does not exhibit a clear external sexual dimorphism as found in the cyprinodontid and poecilid fishes. However, Banarescu and Nalbant (1964) report that pectoral fin rays 2-5 are widened and thickened in males of Persian loach. Also according to Coad (2016), the males have numerous fine tubercles on the dorsal surface of their pectoral fin rays in bands similar to our observations. An unequal sex ratio was observed, which may reflect different survival rates for males and females. It appears that the strategy of this species, in terms of the sex ratio, is the "investment" in females. This is brought about through the selective predation of males or the higher survival rate and greater longevity of females, or the greater endurance of females to environmental stress which is reported in other fish species (see Esmaeili and Shiva, 2006). The results of the present study showed that O. persa spawns in the middle of spring. The mean values of gonado-Somatic index, percentage of late gonad maturation stages (IV, V) and high frequency of large oocytes confirmed the spawning season. The season of spring reported as the spawning season for some other loaches. For example, the gonado- somatic index of Paracobitis malapterura indicated that reproduction of this species occurred in April– May (Pattimar et al., 2009). Mousavi-Sabet et al. (2011) also reported the spawning of Cobitis keyvani from Talar River occures from May to late July. The reproductive index of females was higher than males. This matter is justified according to more volume of female’s gonad (Mahomoud et al., 2011). This index has been widely used as indicator of the fish spawning season, but its use in reproductive biology studies is more suitable when it is associated with other reproduction indicators such as macroscopic and histological techniques (Ghasemian et al., 2015). In the present study, based on the size, shape and weight of the gonads, degree of occupation of the body cavity, presence or absence of ripe oocytes or milt, diameter of the oocytes in the ovary, oocyte shape, vitellogenesis, size of oil droplets and yolk vesicles, five stages of ovary development and four stages of testis development were observed. Many studies have been performed on histological and morphological changes of ovary in fishes (Biswas, 1993). Generally, the process of teleost oogenesis may be divided into 5-8 stages (West, 1990; Fishelson et al., 1996; Ünver and Ünver-Saraydin, 2004). The results of the histological study of gonad development in O. persa is basically similar to other teleost. The process of oogenesis has been divided into 5 stages which resemble many species such as Aphanius farsicus (Monsefi et al., 2007). The testes development process of O. persa revealed 4 sexual stages corresponding to A. farsicus and Cyprinus carpio. The oocyte reaches maximum size of 1.11 mm in O. persa, this is directly found that the nutritional state of the fish affects the vitellogenic and maturation of oocyte because there is a close relationship between the vitellogenesis and oocyte size (Wallace and Selman, 1978). The ecological factors, such as temperature, photoperiod and nutrition are important environmental factors for regulations of reproductive pattern in most teleost (De Vlaming, 1972). As conclusion, the Persian loach, O. persa demonstrates some reproductive strategies, including significantly female biased sex ratio, little sexual dimorphism and spring spawning in response to its habitat. The small body size is another significant factor in the life history of O. persa which allows this loach to colonise and exploit microenvironments. However, the limited distribution makes the species highly vulnerable and could result in significant loss if habitats are disturbed or destroyed. 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(2016) 4(1): 31-42 E-ISSN: 2322-5270; P-ISSN: 2383-0956 Journal homepage: www.ij-aquaticbiology.com © 2016 Iranian Society of Ichthyology چکیده فارسی ,Oxynoemacheilus persa (Heckel پارسی ماهیلوچ در مثلیتوليد نمایه و اگناده بافتی -ریختی هایویژگی ایران کر، رودخانه حوضه از( خار بدون جویباری ماهيانلوچ: عالی استخوانی ماهيان) ،(1847 الفقهایی نخبه محسن ،*اسماعيلی حميدرضا ميرغياثی، سرور .ايران شیراز، شیراز، دانشگاه علوم، دانشکده شناسی،زيست بخش تکوينی، سیستماتیک و شناسیماهی آزمايشگاه چکيده: Oxynoemacheilus persa پارسی ماهی لوچ ماده و نر گنادهاي جنسی بلوغ مراحل و تولیدمثلی نمايه بافتی،-ريختی هايويژگی مطالعه، اين در (Heckel, 1847)، در و انجام الکتريکی صید وسیلههب ،0200 آوريل تا 0202 آوريل ماه از بردارينمونه. است گرديده ارائه ،ايران بومزاد گونه يک عرض و طول وزن، گیرياندازه ريختی، بررسی از پس و گرديده جنسیت تعیین خارج، بدن از گنادها. گرديد آوري جمع ماهیقطعه 091 تعداد کل رب. گرديد آماده شناسیبافت مقاطع ائوزين، و هماتوکسیلین آمیزيرنگ معمول روش از استفاده با و تثبیتدرصد 02 فرمالین محلول در ها،آن رد میلت رنگ شیري مايع يا و ماده گناد در تخمک وجود عدم يا و وجود شکمی، حفره طولبه نسبت گناد اندازه گنادها، وزن و شکل اندازه، اساس مراحل بررسی نتايج. گرديد منظور نرها براي مرحله 4 و هاماده براي جنسی بلوغ مرحله 5 شناسی،بافت مشاهدات و تخمدان در تخمک قطر نر، گناد -يگناد میانه میزان با جنسی بلوغ مراحل و نموده ريزيتخم بهار میانه در ماهی اين که داد نشان پارسی ماهی لوچ ماده و نر گنادهاي جنسی بلوغ .است مرتبط بدنی .بدنی-گنادي نمايه جنسی، بلوغ گناد، شناسیبافت گناد، شناسیريخت تولیدمثل، :کلمات کليدی