Chondrostoma Regium) (Teleostei: Cyprinidae) in Bibi-Sayyedan River, Tigris Basin

BIHAREAN BIOLOGIST 15 (1): 25-32 ©Biharean Biologist, Oradea, Romania, 2021 Article No.: e201304 http://biozoojournals.ro/bihbiol/index.html

Reproductive biology and gonad histology of King Nase (Chondrostoma regium) (Teleostei: Cyprinidae) in Bibi-Sayyedan River, Tigris Basin

Fatemeh KIANI, Yazdan KEIVANY* and Fatemeh PAYKAN-HEYRATI

Department of Natural Resources (Fisheries Division), Isfahan University of Technology, Isfahan 84156-83111, Iran. *Corresponding authors, Y. Keivany, E-mail: [email protected]

Received: 14. February 2020 / Accepted: 01. August 2020 / Available online: 12. August 2020 / Printed: June 2021

Abstract. The reproductive characteristics of Chondrostoma regium in Bibi-Sayyedan River, Iran, were investigated by monthly collections from March 2010 to March 2011. The 471 collected specimens were transferred to the laboratory on ice and biometric measurements were carried out. For the histological studies, gonads were fixed in 10% formalin, hydrated and casted in paraffin, then sectioned transversely at 5-7 µm thickness and stained with Hematoxylin–Eosin. The sex ratio was 1M:2.3F. Age groups of the males and females were 1-4 and 1-5 years. The maximum length (TL) and weight of males was 18.1 cm and 65 g and those of females were 22 cm and 91 g. The minimum and maximum absolute fecundity was 600 and 6500 (3300±200). The average egg diameter was 0.65±0.01 (±SE) mm, the minimum and maximum egg diameter was 0.27 and 1.70 mm. The mean of gonadosomatic index (GSI) was 1.53±0.1 in males and 4.37±0.24 g in females. The GSI showed significant differences in different months in both males and females. The maximum value of GSI in females was observed in March and was significantly different from that of other months. According to histological studies, five sexual maturity stages including: immature, in maturing, ripening, ripe and spent, were observed. Based on GSI values, egg diameter and histological studies, it can be expressed that this species has a short spawning period during late March to early April. Our results indicated that the ovary in king nase is group synchronous.

Key words: King Nase, age, histology, GSI, fecundity.

Introduction In this study, 471 specimens were captured by monthly collections from March 2010 to March 2011. Specimens were caught by electro- King Nase (Nazok or Nazak in Farsi) is a bentopelagic cy- fishing, seine net and cast net, anesthetized and transported to the laboratory on ice and subjected to dissection and biometric meas- prinid fish widely distributed in Iran and adjacent countries urements (length to the nearest 0.1 cm, and weight to the nearest 0.01 and inhabits both lentic and lotic waters (Gumus et al. 2002). g). For each specimen, 10-15 scales were removed from above the This species is found in Tigris (Karun and Karkheh river ba- lateral line below the anterior extent of the dorsal fin on the left side sins) of Iran (Keivany et al. 2016, Esmaeili et al. 2017, Coad of the fish, washed in water and dried between two slides for micro- 2018), however, the reproductive biology of this species is scopic studies (Lagler 1956, Chugunova 1959). Scales, mounted dry not well documented in Iran and other countries, partly be- between glass slides, were used for age estimation and were read by cause this fish is not of commercial importance (Soofiani et three people. Opercles and otoliths were used for age validation. Sex al. 2014, Kiani et al. 2016). Nevertheless, it is used as an edi- was determined by macroscopic and microscopic examination of the gonads. The sex ratio deviation from 1:1 was tested statistically by ble fish at a local scale, but there is no information on its chi-square analysis (Sokal & Rohlf 2012). X2= ∑(Oi-Ei)2/Ei landings. The relative gonad weight or gonadosomatic index (GSI) of fe- Success of reproduction depends upon normal gonadal males and males were calculated as the proportion of the weight of development stimulated by favorable environmental condi- the gonad to the weight of eviscerated body in percent (King 1995). tions (Lam 1983, Shankar et al. 2007, Hontela & Stacey 1990). Also, water temperature was measured to determine the relationship Reproductive development in fishes is well understood by between temperature and gonad growth by a digital thermometer. histological techniques which provide reliable information The absolute fecundity was determined by the gravimetric method after keeping the eggs in Gilson solution for two days. Three sub- on the reproductive state of fishes (West 1990). The ovarian samples were taken from different parts of the ovaries (Biswas 1993). histological pattern of teleosts is described according to the F=n G/g division of ovarian tissues into different groups by different While relative fecundity was determined as the proportion of authors. Recently, Brown-Peterson et al. (2011) provided a absolute fecundity to the eviscerated body weight (number of ma- standardized five stage terminology for describing repro- ture eggs per gram of the body weight) in stage IV of maturation. ductive development in fishes based on the presence of Egg diameter was measured with an ocular micrometer. The diame- dominant gametogenic cell types. ter of 250-300 eggs was measured every month at X40 (Hopkins

The aim of this study was to investigate the reproductive 1979). R= F/Tw The ovaries and testes were processed according to standard his- behavior and processes of C. regium in Bibi-Sayyedan River tological techniques (Humason 1979). All tissue blocks were sec- of Semirom using visual observation and gonadal histology. tioned at 5-6 μm and stained with hematoxylin and eosin. Histologi- The results of this study could be used for management cal slides were viewed under a light microscope. Maturity stages purposes of this species. were determined following Brown-Peterson et al. (2011). Reproduc- tive seasonality was determined by examination of the monthly changes in the gonadosomatic index and the water temperature was Materials and methods recorded during the sampling (10.5 to 21.4C°), in Bibi-Sayyedan Riv- er. The hepatosomatic index was calculated as HSI= (HW/TW)×100. Bibi-Sayyedan River, a water body located approximately 18 km One-way ANOVA was used to analyze differences in means of GSI% south of the town of Semirom (31°10’48”E, 51°24’00”N, 1300 m eleva- and egg diameter of fish at 95% probability level followed by Dun- tion), flowing south-westward from the high central part of Zagros can post hoc test in SPSS (vers.18). Mountain and finally joining Karun River, a tributary of Tigris basin.

26 F. Kiani et al.

Table 1. Monthly variations in the frequency, total length (cm) and total weight (g) of C. regium.

Mean Total Mean Total Months n TL Range Weight range Length±SE Weight±SE March 2010 50 5.5-13.8 7.73 ± 0.31f 1.43-26.35 5.29 ± 0.88d April 44 5.6-15.2 8.01 ± 0.35f 1.60-30.03 6.20 ± 1.02d May 27 6.4-16.5 9.60 ± 0.51 2.75-45.03 10.78 ± 1.95 June 40 2.7-16.0 10.04 ± 0.40 2.82-44.39 11.02 ± 1.61 July 23 7.1-15.4 9.78 ± 0.41e 3.7-33.97 9.38 ± 1.31cd August 22 9.6-12.9 10.84 ± 0.34de 7.43-22.70 12.71 ± 1.89cd September 26 8.4-13.2 9.83 ± 0.21e 5.64-22.45 9.26 ± 0.71cd October 10 9.5-13.9 11.54 ± 0.48d 6.12-21.37 12.40 ± 1.70cd November 08 10.3-17.3 12.07 ± 0.80dc 9.76-49.99 16.12 ± 4.85bc December 14 6.0-12.5 9.62 ± 0.65e 1.95-17.65 10.07 ± 1.53cd January 41 8.8-15.9 13.22 ± 0.29bc 5.58-36.78 21.79 ± 1.49b February 96 9.8-19.3 14.83 ± 0.20a 6.7-76.11 32.73 ± 1.43a March 2011 70 10.6-21.5 13.93 ± 0.26ab 10.8-90.53 29.95 ± 1.77a

Results

The length and weight of females ranged from 5.5 to 21.5 cm (11.29±0.19) and 1.43 to 90.53 (17.52±0.87) g and those of males from 6.0 to 18.1 cm (12.30±0.267) and 1.87 to 64.93 (20.91±1.18) g, respectively (Table 1). The specimens were composed of 320 (50%) females, 138(32%) males and 13 (18%) immature (Fig. 1). The ratio of male to female was 1M:2.3F which was significantly different from the expected 1:1 ratio (χ2= 16.866, p<0.01) (Tables 2 & 3). The oldest males and females were 4 and 5 years old, respectively, with dom- Figure 1. Monthly gonadosomatic index of male (top) and female C. inance of age group 3 in both sexes. regium (bottom) in Bibi-Sayyedan River. Significant differences were found in the males and females GSI (p<0.05) in different months. GSI started to increase in July to February and significantly increased in Table 2. Sex ratios of C. regium in Bibi-Sayyedan River in different months. March, and significantly decreased in May. The highest GSI value for both sexes occurred in April and then started to Months Females Males Sex ratio M/F X2 decrease (Fig. 1). Thus, the reproductive period for this spe- March 2010 32 15 0.46 06.14* cies in this particular environment is from March-April April 35 7 0.20 18.66* when GSI is considerably higher. The lowest hepatosomatic May 25 2 0.08 19.59* index was in July and the highest in November, with a nega- June 30 5 0.16 17.85* tive correlation with GSI (Fig. 2). July 19 4 0.21 09.78* The absolute fecundity of 66 females determined during August 12 9 0.750 00.43 the spawning period was 600-6600 (3300±200) eggs and rela- September 14 12 0.85 00.15 tive fecundity was 30-260 (100±5) eggs/g of body weight. October 7 3 0.42 01.60 The fecundity-age, length and weigh relationships were as November 5 3 0.60 00.50 * F= 272.5A2.182 (r2= 0.77), F= 116.8W-2.182 (r2= 0.71), F= 272.5L- December 11 2 0.18 06.23 7740 (r2= 0.71), respectively (Fig. 3-5). The egg diameter January 23 18 0.78 00.61 * ranged between 0.27-1.70 (0.65±0.01) mm and was signifi- February 64 31 0.48 11.46 cantly different during the year (P<0.05). The egg diameter March 2011 43 27 0.62 03.65 started to increase in July and reached the highest in March. *Significant at 95% confidence limit Obviously, the highest mean egg diameter (0.745±0.06 mm) was observed in March and the lowest in September (0.336 ± Table 3. Sex ratios in different ages of C. regium in 0.011 mm) (Fig. 6). The highest egg diameter in April was Bibi-Sayyedan River. 2.25 mm. Age Males Females Sex ratio Significance The gonad development stages of C. regium are de- 1 22 90 0.24 P<0.05 scribed in five stages following Brown-Peterson et al. (2011) 2 40 96 0.41 P<0.05 which has been accepted by many researchers as the stand- 3 60 105 0.51 P<0.05 ard procedure (e.g., Tomkiewicz et al. 2011, Grier 2012, 4 16 26 0.61 p>0.05 Abbaszadeh et al. 2013, Dopeikar et al. 2016, Hamzeh et al. 2017, Keivany et al. 2017, 2018, Siami et al. 2017). Stage I (never spawned). Testes and ovaries were very transparent grayish in color, blood vessels indistinct. Eggs small and near the spinal column, often clear, threadlike and were not visible to the naked eye and sex could not be dis- Reproductive biology of Chondrostoma regium in Bibi-Sayyedan River 27

Figure 2. Monthly gonadosomatic index and hepatosomatic index of Figure 3. Absolute fecundity-age relationships in C. regium in Bibi- female C. regium in Bibi-Sayyedan River. Sayyedan River.

In the testes (spermatogoni stage), seminiferous tubules with low value and only spermatogonia (Sg) were present; no lumen in lobules. This stage was observed in May. Stage II (Early developing; developing; ovaries and testes beginning to develop, but not ready to spawn). Ovaries and testes like an opaque grey-smoky tube occupying almost half of the body cavity. Blood capillaries were evident on the surface. Cytoplasm thickened (cytoplasmic/prenuclear stage). Nucleus large and global containing many nucleulii

that migrate around the nucleus wall making a regular chain Figure 4. Absolute fecundity-weight relationships in C. regium in Bi- around it; some remain in the middle. Immature eggs which bi-Sayyedan River. resemble white spots were visible to the naked eye. Primary

growth, cortical alveolar (CA), primary vitellogenic (Vtg1), and secondary vitellogenic (Vtg2) oocytes were present. Like the previous stage, oocytes are irregular and polygon in shape with visible yok granules in their cytoplasm. Oocytes surrounded by follicles. Early developing subphase: only PG and CA oocytes present (Fig. 7B). The mean diameter of oocyte was 12.00±01 µm. Some remains of a few oogonia were present. This stage is also called regeneration since after stage V, mature ovaries return back to this stage to resume oogenesis. Testes were small but easily identiﬁable (spermatogenesis stage). Testes, an opaque light greyish tube, occupying almost half of the body cavity. Blood capillaries were evident on the surface. Spermatocysts were evident along the lobules. Primary spermatocyte (Sc1) and secondary spermatocyte (Sc2) were present in spermatocysts (Fig. 8B). This stage was observed during June-September. Stage III (Spawning capable; ﬁsh were developmentally and physiologically able to spawn). Gonads filled the body

Figure 5. Absolute fecundity-length relationships in C. regium in Bi- bi-Sayyedan River.

tinguished (Fig. 7A). Primordial germinal cells were modify- ing to oogonia. Oogonia is a polygon with a large and global nucleus and chromatin. A thin and basophil cytoplasm sur- rounding the nucleus (purpled by hematoxylin-eosin). The mean diameter of oocytes was 7±0.5(SD) µm. In histological sections of ovaries, only oogonia and primary growth (PG) oocytes were present. No atresia. Ovarian walls were thin Figure 6. Monthly variations (±SE) in egg diameter of C. regium in Bibi-Sayyedan River.

and there was little space between oocytes. 28 F. Kiani et al.

Figure 7. Histological stages of C. regium ovarian development stained with hematoxylin and eosin. A) Stage I (Immature X10). B) Stage II (Developing X10). C) Stage II (Developing X4). D) Stage III (Spawning capable X10). E) stage Iv X4 F) Stage IV (Regressing X10). Ca: Corticule alveoli, Cy: cytoplasm, Io: immature oocyte, N: nucleus, Ne: nucleolus, Pn: Prenuclear, POF: Empty postovulatory follicle, Yg: Yolk vesicules, Zr: Zonata radiata.

cavity. Ovaries were orange due to development of the hydration stage, yolk globules filled the entire cytoplasm. blood vessels (vitellogenic stage). Migratory nucleus and Nucleus was observed at animal pole (Fig. 7C,D). Mean di- hydration stages were distinguished. In migratory nucleus ameter of oocytes was 32.00±10 µm. The highest diameter stage, nucleus began to leave central position and migrate was observed in this stage (2.25mm). Blood vessels were ev- towards periphery. Yolk globules filled more than two third ident on the surface. Oocytes absorbed water, follicular epi- of the cytoplasm. Oocyte size remained relatively stable. In thelium broken, intercellular space increases and yolk glo-

Reproductive biology of Chondrostoma regium in Bibi-Sayyedan River 29

Figure 8. Histological stages of C. regium testicular development stained with hematoxylin and eosin (X40). A) Stage II (Develop- ing). B) Stage III (Spawning capable). C) Stage IV (X40) D) Stage IV (X40). Sc1: primary spermatocytes, Sc2: Secondary spermatocytes, Sd: Spermatids, Sg: Spermatogonia, St: spermatid.

bules occupied all over the cytoplasm. Stage V (Regenerating; sexually mature, reproductively Testes were large and ﬁrm and whitish in color (spermi- inactive). Ovaries and testes were empty, loose and red. Few ogenesis stage). Some sperms were released after applying remains of eggs in resorption process (Artesia) (Fig. 7). Gon- hand pressure. Primary and secondary spermatocytes, ads return to stage II of maturity for the next spawning sea- spermatids and a few spermatogonia were present. Sperma- son. This stage was observed in March and April. tozoa were observed in lumen of lobules and/or sperm In the final two stages or after ovulation, the spent ova- ducts. Spermatocysts were present throughout the testis and ries were small, bloodshot and granular with scattered re- spermatogenes were active (Fig. 8C). Actively spawning sidual oocytes. These ovaries composed of postovulatory fol- subphase (macroscopic): milt released with gentle pressure licles, immature oocytes and mature eggs left unspawned. on abdomen. This stage was observed in January to March. Ovary is a synchronous type. The spawning period of C. re- Stage IV (Regressing; cessation of spawning). Ovaries gium in Bibi-Sayyedan River was determined based on the were flaccid and blood vessels prominent. Atresia (any GSI (Fig. 1), analysis of seasonal development in mean egg stage) and POFs were present. Some CA and/or vitellogenic diameter (Fig. 2), and direct observation of the gonads. The (Vtg1, Vtg2) oocytes were present (Fig. 7E, F). Testes were lowest temperature was in March and then began to increase small and ﬂaccid, no milt released with pressure. This stage in April along with daytime and a sudden increase in river was observed in January to March. flooding. Therefore, based on macroscopic, microscopic and 30 F. Kiani et al. environmental variations, the spawning period continued cided with GSI (Wootton 1984). The lowest HSI was ob- approximately from March to May when water temperature served in June that may be due to smaller size of the speci- was between 17 and 20°C. mens, more immature specimens and hormonal variations. Fecundity in females was not highly correlated with total length and total weight. These patterns were not similar to Discussion other barbs. The possible explanation for this phenomenon is that the smaller fish produced more but smaller eggs, The abundance of males was lower than females. In Euphra- whereas larger fish produced fewer but larger eggs. The egg tes River this ratio was reported as 1M:1F (Sevik 1997), in diameter in this study was lower than that reported from Ataturk Dam as 1M:0.7F (Oymak 2000), in Karakaya Dam as Sour River (Balci et al. 1990) and Kuban Dam (Coban & Sen 1M:1.2F (Kalkan & Erdemli 2003), Sour River as 1:2 (Erguden 2006). et al. 2010), Sair Dam 1:1.2 (Kara & Solak 2004), Seyhan Dam This work represents the first attempt to study the his- 1:1.2 (Suicmez et al. 2011), Almus River as 1:066. This might tology of oocyte development in C. regium. In most cases, the be due to the differential occurrence of males and females in resting, previtellogenic and vitellogenic stages were visible various water columns or that females live in deeper areas from spawning season to next March. Also, in most speci- which make them less available or naturally, the abundance mens collected in April, the main phase of nuclear migration of females is less than that of males in this population, or and hydration, and in some instances, wrinkled follicles might be due to different behaviors in the two sexes leading were visible; however, the maximum weight of gonads and to an easier catch of one sex (Rajaura 1992). Also, differences GSI and maximum diameter of the eggs were seen in the in mortality of the sexes (Sandovy et al. 1994). Differences in same month. Hydration stage in fish is known as an indica- fishing methods and equipment could also be a possible rea- tor of readiness for spawning (Ganias et al. 2004). During the son. It also may differ from one population to another and rest period, cells had a large and single nucleus with a high from one year to another in the same population (Nikolsky nucleus to cytoplasm ratio. The nucleus contained one to 1963). The low proportion of higher age classes suggests that four nucleoli and cytoplasm was strongly basophilic, but the very few individuals survive to a maximum age, as is typical number and size of the nucleolus increased in perinucleolar of most fishes (Matthews 1998). The dominance of younger stage and also, had migrated to the nuclear margin. Based on individuals implies that there might be a fisheries or preda- the formations of ovarian follicles, the first stage of oocytes tor pressure or pollution effect on the population (Yapici et growth occured during peripheral nucleolus stage, this is al. 2012). exactly when the oocytes were covered by the follicular cells. In this study, the maturity age was estimated as 1 year at Hepatic yolk uptake and storage of the oocyte cyto- 4.5 cm for males and 2 years for females at 5.5 cm. Balci et al. plasm, marks the beginning of vitellogenesis. Main oocyte (1990( reported the minimum maturity age as 3 years in Sour growth occurs in the vitellogenesis phase (Hibiya et al. 1982). River. In Euphrates, the age at first spawning of C. regium With the beginning of the vitellogenic yolk, vesicles are visi- was 3 years in males and 4 years in females (Sevik 1997). In ble in the marginal cytoplasm and toward the end of the vi- Ataturk River, it was 2 in males and 3 in females (Oymak tellogenesis; they increase in number and size. It should be 2000) and in Sair Dam, 2 years in both sexes. In Kaban dam noted that during the process of oocyte growth, cortical al- age at first maturity was 2 in males and 3 in females (Coban veolar (simultaneously with the appearance of yolk vesicles), & Sen 2006). The first spawning age is affected by species, thecal, granulose and zona radiata layers gradually make size of fish and environmental factors such as temperature, their appearance and thickness of these layers is increased quality of food and feeding (Yildirim et al. 2001, Schultz et with oocyte growth. Seemingly, thecal and granulosa layers al. 1996, Hoenig & Hewitt 2005). are contributing in making ovarian steroids (Hibiya et al. From the maximum recorded GSI values, it is evident 1982). Macroscopic observations of larger fish gonads in that the reproductive season of C. regium was from late winter (coinciding with the period of vitellogenesis) showed March to early April with a short spawning period. In that ovaries were yellowish to non-transparent, with appar- Ataturk River, was during April-July (Oymak 2000), in Sair ent capillaries. It was observed that during this period, the Dam during April-June and in Kaban dam during February– number of yolk vesicles toward the cell center are now fac- June (Coban & Sen 2006). Nikolsky (1963) pointed out that ing up to occupy the total space of the cytoplasm. Similar the spawning characteristics of a fish vary in respect to their findings were observed in other cyprinids (Unver & Saraya- species and ecological characteristics of water systems in din 2004, Asadollah et al. 2013, Dopeikar et al. 2015). which it lives. During maturation period in March, ovaries were or- The maximum fecundity in C. regium was 6500. Balci et ange, lobular, and with visible capillaries in surface occupy- al. (1990) reported it as 11300, Oymak (2000) in Ataturk Dam ing the whole coelom. In this phase, nuclei migration toward as 15500, Coban and Sen (2006) in Kuban Dam as 16800. It is the animal pole of oocytes had begun and more hydrated well known that fecundity is affected by age, size, species, oocyte was visible. Nuclear membrane disappeared in this feeding, season and environmental conditions (Das 1977). stage. The follicles were the thickest at this time. The GSI Additionally, it differs among populations of some species peak and maximum diameter of the eggs was also observed and does not remain constant from year to year (Bhuiyan et in March. In some specimens, Dopeikar et al. (2015) ob- al. 2006, Coban & Sen 2006). served similar things in Barbus lacerta in the same river. Pres- There is a negative correlation between GSI and HSI ence of empty follicles indicates that the fish spawns in mid since the liver fat used for yolk production in oocytes. In to late March as the temperature increases (Unver & Saraya- most fishes, HSI lower peak occurs slightly before or coin- din 2004). 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