Reproductive seasonality and maturity of male Neophocaena phocaenoides asiaeorientalis in captivity: A case study based on the hormone evidence

To provide hormone evidence on reproductive seasonality and maturity of the Yangtze finless porpoise, the authors monitored the monthly variations of serum reproductive hormones of a male Yangtze finless porpoise in captivity via radioimmunoassay from 1997 to 2003. It was demonstrated that the immature animal had a serum T level of 28-101 ng dL^-1 (65 ± 52 ng dL^-1), reached puberty with the serum T concentration ranging between 250 and 590 ng dL^-1 (390 ± 130 ng dL^-1), and attained reproductive maturity when the serum T level went beyond 1120 ± 180 ng dL^-1 in the breeding season. The body length growth of the animal showed a pattern similar to the serum T variations during its adolescent period from the age of 4 to 6 years. Moreover, the serum T concentration of the male porpoise exhibited significant seasonal variations, indicating that its breeding season may start as early as March and end as late as September.     

Serum concentrations of gonadotropins and steroid hormones of Neophocaena phocaenoides asiaeorientalis in middle and lower regions of the Yangtze River

As a relatively isolated and unique freshwater population, the Yangtze finless porpoise (Neophocaena phocaenoides asiaeorientalis) is the most endangered subpopulation of this species. The objective of this study was to improve our understanding of their reproductive endocrinology by measuring (radioimmunoassay) serum concentrations of FSH, LH, estradiol (E(2)), progesterone (P(4)) and testosterone (T(2)) in free-ranging animals. Blood samples were collected from 66 Yangtze finless porpoises (41 males and 25 females) captured in the middle and lower reaches of the Yangtze River. Based on significant variation of serum T(2) concentrations in males with body length (BL) >138 cm, we inferred that they were mature; in these animals, there were significant seasonal variations in serum T(2) concentrations, with the highest concentrations in March and April (502.0+/-319.8 ng/dL, mean+/-S.D.) and the lowest in December (79.4+/-83.2 ng/dL). Serum T(2) concentrations positively correlated with serum concentrations of LH and weakly correlated with serum concentrations of E(2), whereas there was a significant negative correlation between serum LH and FSH concentrations in males >138 cm. The smallest apparently pregnant female porpoise had a BL of 130 cm. Serum P(4) concentrations ranged from 13.2 to 112.4 ng/mL (43.9+/-28.3 ng/mL) in pregnant females, and fluctuated under 1.0 ng/mL in non-pregnant females with BL>130 cm. Serum LH concentrations were significantly higher in non-pregnant females >130 cm versus those females <130 cm. To our knowledge, this is the first study of endocrine-related maturity and seasonal breeding characteristics of the Yangtze finless porpoise.     

长江江豚繁殖生物学研究概述

长江江豚,即江豚扬子亚种,是仅分布于长江及附属湖泊中的唯一江豚淡水亚种,也是3个江豚亚种中最濒危的一个亚种。本文概述了30 年来国内外关于长江江豚繁殖生物学方面的研究成果,主要从繁殖生态学、组织解剖学、生理学以及行为学等4 个方面阐述了长江江豚繁殖生物学研究现状,并对其中一些相互矛盾和有争议的问题进行了分析。探讨了今后研究中应注意的重点问题,力图为长江江豚的繁殖生物学研究指明方向,为早日实现长江江豚的人工繁殖奠定基础。
     

Establishment and characterization of fibroblast cell lines from the skin of the Yangtze finless porpoise

The Yangtze finless porpoise (Neophocaena phocaenoides asiaeorientalis), as the sole freshwater subspecies of N. phocaenoides, is endemic to the Yangtze River and its adjacent lakes. Its population has declined significantly over recent decades. In this study, we established a skin-derived finite fibroblast cell line of the Yangtze finless porpoise, named YFP-SF1, using primary cell culture methods, and an immortalized cell line, T-YFP-SF1, through co-transfection (GFP and SV40 T antigens) techniques. YFP-SF1 proliferated continuously with a minimum population doubling time of 31 h and exhibited age-dependent changes in growth rate. T-YFP-SF1 cells exhibited fibroblast morphology and were characterized by a shorter doubling time, higher attachment efficiencies, colony formation at a low seeding density, and growth in low serum concentrations. Anchorage independence and foci formation in the cell monolayer were observed from passage 36. The chromosome number of YFP-SF1 and T-YFP-SF1 remained stable at 2n = 44 in the early passages, and the viability of thawed cells remained above 90% after cryopreservation in liquid nitrogen. Taken together, we have established fibroblast cell lines of Yangtze finless porpoise for the first time, which might assist as an in vitro model for this endangered mammal.     

The reproductive cycle of female Ballan wrasse Labrus bergylta in high latitude,temperate waters

This 2 year study examined the reproductive cycle of wild female Ballan wrasse Labrus bergylta in western Norway as a precursor to captive breeding trials. Light microscopy of ovarian histology was used to stage gonad maturity and enzyme‐linked immuno‐absorbent assay (ELISA) to measure plasma concentrations of the sex steroids testosterone (T) and 17β‐oestradiol (E₂). Ovarian recrudescence began in late autumn to early winter with the growth of previtellogenic oocytes and the formation of cortical alveoli. Vitellogenic oocytes developed from January to June and ovaries containing postovulatory follicles (POF) were present between May and June. These POF occurred simultaneously among other late maturity stage oocytes. Plasma steroid concentration and organo‐somatic indices increased over winter and spring. Maximal (mean ±s.e .) values of plasma T (0·95 ± 0·26 ng ml^?1), E₂ (1·75 ± 0·43 ng ml^?1) and gonado‐somatic index (I_G; 10·71 ± 0·81) occurred in April and May and decreased greatly in July when only postspawned fish with atretic ovaries occurred. Evidence indicates that L. bergylta are group‐synchronous multiple spawners with spawning occurring in spring and peaking in May. A short resting period may occur between late summer and autumn when previtellogenic oocytes predominate and steroid levels are minimal.     

Spermatozoa motility and variation in the seminal plasma proteome of Eurasian perch (Perca fluviatilis) during the reproductive season

This study evaluated physiological and functional sperm parameters and the seminal plasma proteome of Eurasian perch (Perca fluviatilis) over the course of their reproductive season. Spermatozoa velocity (169.56 ± 6.53 to 158.5 ± 7.4 µm sec^?1), percent motility (95.89 ± 4.28% to 89.55 ± 4.5%), and osmolality of seminal plasma (290 ± 5 to 297 ± 12 mOsmol kg^?1) remained stable throughout the reproductive season. Milt volume and protein concentration of seminal plasma gradually increased and reached the highest values late in the reproductive period. Spermatozoa concentration peaked in the mid‐reproductive season (66.90 ± 13 × 10⁹ spermatozoa ml^?1) and decreased towards the end (54 ± 10 × 10⁹ spermatozoa ml^?1). A proteomic analysis of seminal plasma using two‐dimensional polyacrylamide gel electrophoresis revealed 10 protein spots significantly altered over the course of the reproductive season. Subsequent protein characterization suggested that time in the reproductive season predominantly affected proteins involved in membrane trafficking, organization, cell motility, and oxido‐reductase activity. This study provides new data on physiological properties of sperm and protein patterns of seminal plasma over the course of the reproductive season that should be considered in the development of methods for artificial reproduction of perch. Mol. Reprod. Dev. 79: 879–887, 2012. © 2012 Wiley Periodicals, Inc.     

Seasonal changes of progesterone and testosterone concentrations throughout gonad maturation stages of the Mexican octopus,Octopus maya (Octopodidae: Octopus)

Variations in progesterone (P₄) and testosterone (T) levels in the gonad of Octopus maya from Sisal in Yucatan State, Mexico, were investigated by radioimmunoassays and in relation to four gonad maturation stages (GMS) and to the reproductive cycle, as represented by two maturity indices (microscopic ‘MiMI’ and macroscopic ‘MaMI’). According to the GMS and the maturity indices, the reproductive season of O. maya from Yucatan occurred from February to June. In females, P₄ and T displayed the same pattern, with a tendency to increase at the same time, although on average, P₄ had seven-fold higher concentrations than T. In contrast, P₄ and T in male gonads displayed a different pattern, where T concentrations were relatively stable throughout all of the study months. In the female gonad P₄ was lowest (close to 0 pg?g^–1) during both developing (GMS-I) and maturing (GMS-II) stages, and increased (189?±?53?pg?g^–1) approaching the mature stage (GMS-III) to a maximum value of 611?pg?g^–1. Concentrations of T in the male gonad were lowest (106?±?9?pg?g^–1) during the maturing stage (GMS-II) and increased up to the mature stage (GMS-III), reaching a maximum of 440?pg?g^–1. Pearson's correlation (r) between hormones and maturity indices showed strong relationships for females (around 0.4 and ?0.7; p?<?0.05), but there were negligible or weak relationships for males (0.2 and ?0.1; p?>?0.05). Hormone correlations in females were inverse with MaMI and direct with MiMI. Our major findings showed that gonadal P₄ levels were elevated during GMS-III and GMS-IV (i.e. periods of vitellogenic oocytes), where the characteristic aspect is an ovary with very high oocyte diameters, with the primary follicle cells deeply infolded in the ooplasm for yolk synthesis. These results suggested a synchrony between P₄ and the process of folliculogenesis, and in turn, vitellogenesis.     

Seasonal changes in the local distribution of Yangtze finless porpoises related to fish presence

The Yangtze finless porpoise (Neophocaena asiaeorientalis asiaeorientalis) is an endangered freshwater porpoise subspecies unique to the Yangtze River basin. Seasonal variations in local distribution of the animal, as well as fish presence, sand dredging, ship navigation, and bridges were examined as potential factors affecting the occurrence of the animals. Passive acoustic surveys were performed regularly from May 2007 to August 2010, near the conjunction of the Yangtze River and Poyang Lake. The distribution of the porpoises was seasonally site‐specific. In May and August, the animals were detected more often at river junctions than in the lake, but vice versa from November to February. The rate of the porpoise detection was significantly higher in areas of fish presence than in areas of absence. The number of porpoises detected did not differ significantly between the sand dredging operation and the prohibition period (in 2008), although the number of vessels obviously declined in 2008. Ship traffic and bridges also did not appear to affect the presence of porpoises. These results showed the relative importance of the various environmental factors, which is important for conservation of not only Yangtze finless porpoise but also endangered isolated cetaceans.     

Seasonal changes in the concentration of estrogens and testosterone in the plasma of the stallion

A blood sample was taken from each of 15 stallions at monthly intervals for 14 consecutive months. Plasma concentrations of estrogens and testosterone were measured by radioimmunoassay methods. Estrogens in peripheral blood were present in much higher amounts than testosterone and were principally in a water-soluble, solvolyzable form (> 98%). The major component in the solvolyzed extracts behaved chromatographically as estrone. The mean plasma level (± S.E.) of estrogens averaged across months was 52.9 ± 4.5 ng ml^?1. Individual stallions showed considerable month-to-month variation; for example, single monthly samples ranged from 29.5 to 160.6 ng ml^?1 for the stallion with the highest single value.The highest mean monthly concentration was 69 ± ng ml^?1 in May, and plasma levels were < 40 ng ml^?1 during November and December. For the 11 Thoroughbred stallions in the study, the mean concentrations of estrogens were 73 ± 5.8 ng ml^?1 for May to July and 45 ± 4.1 ng ml^?1 for November to January (P > 0.001).The mean monthly concentrations (± S.E.) of testosterone ranged from 0.22 ± 0.05 to 0.90 ± 0.14 ng ml^?1, and individual samples ranged from < 0.02 to 2.8 ng ml^?1 of plasma. While the highest mean level of testosterone was seen in September, there was a significant difference (P < 0.01) between the values in the breeding season (May–July, 0.73 ± 0.07 ng ml^?1) and the non-breeding season (November–January, 0.38 ± 0.08 ng ml^?1). No marked seasonal changes were observed, however, in testosterone levels in several stallions. It was concluded that plasma estrogen levels may provide a more sensitive index of endocrine function of the testes in the stallion.     

固化河岸对长江江豚栖息活动的影响

长江江豚主要栖居在近岸浅水水域,喜好泥沙质类型的河岸。但在长江干流,有很多自然河岸被固化,河岸固化给长江江豚分布和栖息活动造的影响仍不甚清楚。2016年3月—2017年1月,对长江干流2个自然河段江豚的数量和分布做了12次考察,并收集了这2个河段岸型的相关数据,来分析固化河岸对江豚栖息活动的影响。12次考察累计发现江豚215头次,平均每次考察观察到江豚(17.92±7.09)头次。研究区域的固化河岸约占岸线总长的59%,分析发现,仅约13.9%的江豚分布在固化河岸水域,86.1%的江豚均分布在自然河岸水域。江豚在单位河岸长度的分布数量与该段固化河岸长度所占的比例呈显著负相关(r=-0.639,P0.01)。在自然河岸,分布在近岸50 m水域的江豚占31.8%,而在固化河岸,仅观察到2头江豚活动在近岸50 m水域内。由此可见,固化河岸对江豚的近岸分布有明显影响。调查还发现,安庆城区建设带约10 km江段12次考察均未发现江豚分布,城区建设带可能对江豚栖息已造成严重影响。长江干流的固化河岸所占比例非常高,研究结果提示这可能导致长江干流江豚栖息地的丧失和破碎化加剧,在制定长江江豚保护措施必需慎重考虑此因素的影响,并据此提出相应的栖息地保护和恢复方案。     

长江江豚脐带永生化成纤维细胞系建立及细胞生长特性研究

取长江江豚(Neophocaena phocaenoids asiaeorientalis)产后胎盘脐静脉,经组织块培养,差异贴壁法纯化,构建长江江豚的原代细胞系;经外源癌基因SV40 T antigens(猿猴病毒T抗原)转染构建稳定脐带细胞系,并对长江江豚的永生化后的成纤维细胞的细胞形态、转染效率、生长曲线和活率等...     

Ovarian development and serum sex steroid and vitellogenin profiles in the female cultured sturgeon hybrid, the bester

Changes in serum levels of estradiol-17/J (E₂), testosterone (T), 17,20β-dihydroxy-4-pregnen-3-one (17,20β-P) and vitellogenin (VTG), in cultured adult female bester were examined in relation to ovarian development during a 1-year sampling period. Considerable variations in oocyte development were found among fish. Oocytes at previtellogenic stage (≥0–6mm in diameter) generally started to develop concomitantly with the degeneration of the first batch of oocytes. In vitellogenic individuals, ovaries were comprised of more advanced oocytes with diameter ranging from 0–6 to 2–6 mm and in the post-vitellogenic class, oocytes attained their largest size (>2–6mm) while the germinal vesicle was migrating towards the animal pole. Oocytes with a migrating nucleus were maintained during the winter period and massive degeneration started in April–May without germinal vesicle breakdown (GVBD) or ovulation occurring. Seasonal changes in E₂, T and VTG levels were well correlated with the advancement of oogenesis. Their levels increased during vitellogenesis, whereas in the post-vitellogenic (migratory nucleus) stage the levels of E₂ declined from 2–4 ng ml ^?1± to 1–2 ng ml ^?1 and VTG from 4–10 mg ml ^?1 to 0.–0.5 mg ml ^?1 while T levels remained high (50–60 ng ml ^?1). In contrast, serum levels of 17,20?-P were constantly low (less than 0.2 ng ml^?L) throughout the reproductive cycle. These results indicate that the time appropriate for induction of artificial reproduction would be from October–November to April–May when the oocytes are in the late Stages of the development.     

B-Mode ultrasonographic evaluation of the testis in relation to serum testosterone concentration in male Yangtze finless porpoise (Neophocaena phocaenoides asiaeorientalis) during the breeding season

The use of ultrasonography as a noninvasive tool for assessing the reproductive status of the male Yangtze finless porpoise (YFP; Neophocaena phocaenoides asiaeorientalis) was validated by correlating ultrasonographically determined testicular volume (TV) and testicular parenchyma pixel intensity (PI) with serum testosterone (T) concentration. The testes of 13 free-ranging male YFPs from the Tian-e-Zhou Reserve and three captive animals from the Baiji Dolphinarium (Wuhan, China) were examined ultrasonographically during April 2008. Testis volume was determined using Lambert's formula for an ellipsoid. Testicular parenchyma PI was evaluated by analyzing testicular ultrasonograms using pixel analysis software (Image J). Serum T concentrations were determined using a single-antibody radioimmunoassay. The TV, PI, and serum T concentration were low and similar in animals with body length <133 cm, highest in those with body length ≥142 cm, and highly variable in those with body length from 133 to 141 cm. Both TV and PI were closely correlated with serum T concentration (r = 0.91 and r = 0.85, respectively; P < 0.01), indicating a consistent association between structural and functional development of the testis. In conclusion, we inferred that puberty onset in male YFPs occurred when TV was >150 cm³ and PI was >60 during the breeding season and that testicular ultrasonography and pixel analysis was an efficient, noninvasive, real-time tool to evaluate testicular function of live male YFPs.     

Seasonal changes in serum dehydroepiandrosterone,androstenedione, and testosterone levels in the squirrel monkey (Saimiri boliviensis boliviensis)

The squirrel monkey (Saimiri boliviensis boliviensis) has a well-defined breeding season during which adult males undergo androgen-dependent morphological changes, with acquisition of active spermatogenesis. To assess the hormonal events of this annual cycle, blood samples were obtained weekly from ten adult males, and serum was assayed for testosterone (T), androstenedione (ΔA), and dehydroepiandrosterone (DHEA). A significant seasonal variation was noted in mean serum T (P < 0.02), ΔA (P < 0.02), and DHEA (P < 0.001) concentrations. Mean ΔA concentrations increased from a nonbreeding season nadir of 91.4 ± 12.9 ng/ml (mean ± standard error) to a prebreeding concentration of 139 ± 10.5 ng/ml and breeding season peak of 167.5 ± 15.4 ng/ml (P < 0.05). Mean DHEA concentrations increased from a nonbreeding season nadir of 8.3 ± 0.8 to a breeding season peak of 14.3 ± 1.2 (P < 0.001). Mean T levels in the nonbreeding (52.2 ± 11.6 ng/ ml) and prebreeding season (48.6 ± 7.4) were similar. However, T significantly increased during the breeding season to 103.5 ± 12.8 ng/ml (P < 0.05). Progressive changes in body weight and morphology paralleled the rise in serum ΔA levels. The pattern of peripheral serum androgen concentrations throughout the year would suggest annual activation of the hypothalamic-pituitary-adrenal and/or hypothalamic-pituitary-gonadal axes.     

Population Genetic Structure of the Yangtze Finless Porpoise (Neophocaena phocaenoides asiaeorientalis): Implications for Management and Conservation

Understanding the population genetic structure is a prerequisite for conservation of a species. The degree of genetic variability characteristic of the mitochondrial DNA control region has been widely exploited in studies of population genetic structure and can be useful in identifying meaningful population subdivisions. To estimate the genetic profile of the Yangtze finless porpoise (Neophocaena phocaenoides asiaeorientalis), an endangered freshwater population endemic to China, the complete mtDNA control region was examined in 39 individuals belonging to seven different stocks inhabiting the middle and lower reaches of the Yangtze River. Very low genetic diversity was found (nucleotide diversity 0.0011± 0.0002 and haplotypic diversity 0.65± 0.05). The mtDNA genetic pattern of the Yangtze population appears to indicate a founder event in its evolutionary history and to support the marine origin for this population. Analyses by F_st and Φ_st yielded statistically significant population genetic structure (F_st = 0.44, P < 0.05; Φ_st = 0.36, P < 0.05). These results may have significant implications for the management and conservation of the Yangtze finless porpoise in the future.     

Analysis of myosmine,cotinine and nicotine in human toenail,plasma and saliva

Myosmine is a minor tobacco alkaloid with widespread occurrence in the human diet. Myosmine is genotoxic in human cells and is readily nitrosated and peroxidated yielding reactive intermediates with carcinogenic potential. For biomonitoring of short-term and long-term exposure, analytical methods were established for determination of myosmine together with nicotine and cotinine in plasma, saliva and toenail by gas chromatography–mass spectrometry (GC/MS). Validation of the method with samples of 14 smokers and 10 non-smokers showed smoking-dependent differences of myosmine in toenails (66?±?56 vs 21?±?15?ng?g^?1, p?<0.01) as well as saliva (2.54?±?2.68 vs 0.73?±?0.65?ng ml^?1, p <0.01). However, these differences were much smaller than those with nicotine (1971?±?818 vs 132?±?82?ng g^?1, p <0.0001) and cotinine (1237?±?818 vs <35?ng?g^?1) in toenail and those of cotinine (97.43?±?84.54 vs 1.85?±?4.50?ng ml^?1, p <0.0001) in saliva. These results were confirmed in plasma samples from 84 patients undergoing gastro-oesophageal endoscopy. Differences between 25 smokers and 59 non-smokers are again much lower for myosmine (0.30?±?0.35 vs 0.16?±?0.18?ng?ml^?1, p <0.05) than for cotinine (54.67?±?29.63 vs 0.61?±?1.82?ng ml^?1, p <0.0001). In conclusion, sources other than tobacco contribute considerably to the human body burden of myosmine.     

长江安庆段航道整治对长江江豚数量和分布的时空影响

栖息地丧失和破碎化是威胁长江江豚(Neophocaena asiaeorientalis)种群长期发展的主要因素之一。航道整治工程是导致江豚栖息地丧失和破碎化的一个主要原因,研究工程施工和运行对江豚数量和分布的影响可为江豚栖息地管护提供有益信息。通过对湖口-吉阳矶水域江豚数量和分布的持续调查,分析了安庆段航道整治工程施工和运行对江豚的影响。12次目视调查共在研究水域发现江豚258群次613头次,工程施工前、施工期和初步运行期单次考察分别平均观察到江豚(67.0±24.3)头次、(35.6±22.2)头次和(50.0±2.8)头次。在主要施工区共发现江豚79群次185头次,施工前、施工期和初步运行期单次考察分别平均观察到江豚(18.60±12.03)头次、(10.4±7.3)头次和(20.0±4.2)头次。工程不同时期主要施工区江豚的数量及占比在统计学上没有显著差异。主要施工区S2和S3工程段施工期江豚平均分布密度((0.18±0.17)头/km、(0.04±0.08)头/km)较施工前((0.62±0.16)头/km、(0.40±0.29)头/km)显著下降,S5和S6工程段施工期平均...     

Physiological responses to capture and handling of free-ranging male Yangtze finless porpoises (Neophocaena phocaenoides asiaeorientalis)

We captured free-ranging male Yangtze finless porpoises over three seasons and assayed leukocytes and serum biochemistry to investigate physiological responses to the capture and handlings. Serum thyroid hormones (THs) declined sharply in those porpoises compared with hormone variation in a captive male finless porpoise. Hypernatremia and hypokalemia were also significant in the free-ranging animals suggesting that conservation of serum sodium might be acutely vital for this freshwater subspecies. The animals captured in spring showed more significant neutrophilia and eosinopenia than those captured in autumn suggesting that they may be more affected by capture during the breeding season. Furthermore, physical examination of porpoises when out of the water was apparently stressful, particularly when they were kept out of the water for longer periods. However, an increase in circulating THs may be an adaptive response to accommodate these short-term stresses.     

长江江豚自然保护区建设管理存在的问题及调整建议

长江江豚(Neophocaena asiaeorientalis)是生活在中国长江的珍稀濒危鲸类动物,自然栖息地破坏是造成其种群衰退的重要原因。目前中国已经在长江中下游流域建立了8个长江江豚自然保护区。然而,早期保护区的功能区划随经济发展和生态保护的现实矛盾日渐突出,按照现行法规对保护区难以实行有效管理,迫切需要进行优化调整。文章基于国家政策、长江江豚生物学特点及分布规律,兼顾航运等重大经济发展需求,提出了长江豚类自然保护区调整的原则和建议:(1)将长江江豚喜好的近岸水域及一定范围的重要江滩和洲滩湿地,及江心洲汊江一侧水域划入核心保护区;(2)将中间主航道水域以及长江大堤至核心区边界之间的水域划入一般控制区;(3)兼顾历史遗留问题,细化不同区域的管控措施。该调整方案一方面使保护区功能区划更加符合长江江豚的分布特征和偏好生境,有利于对其自然种群和重要栖息地的有效保护。另一方面也有助于理顺自然保护区管理上存在的法律障碍,为长江江豚自然保护区建设、规划和管理提供新的思路和模式。     

Differences in reproductive strategies between obscure puffer Takifugu obscurus and ocellated puffer Takifugu ocellatus during their spawning migration

Reproductive strategies were compared between obscure puffer Takifugu obscurus and ocellated puffer Takifugu ocellatus captured in waters near Yangzhong Island in the lower reaches of the Yangtze River during the spawning migration season from February to June in two consecutive years (1995 and 1996). Results showed that obscure puffer and ocellated puffer have adopted different reproductive strategies, including different spawning times, different size at maturity, and different number and size of oocytes, resulting in two different larval sizes. In detail, the timing of the spawning migration and status of gonad development of obscure puffer was about 1 month earlier than that of ocellated puffer; the obscure puffer was obviously longer and heavier than ocellated puffer in both mature male and female fish; mean GSI of obscure puffer females (15.8%) was significantly higher than that of ocellated puffer females (14.6%); the average diameter of ocellated puffer eggs (1.49 ± 0.12 mm) was significantly larger than that of the obscure puffer (1.22 ± 0.08 mm); and obscure puffer females (320.8 oocytes mg^?1 somatic wet weight) had significantly higher relative fecundity than ocellated puffer females (125.2 oocytes mg^?1 somatic wet weight). These differences in reproductive strategies between two closely related species of the Takifugu genus indicate that both obscure puffer and ocellated puffer fit the r/K dichotomy. Obscure puffer shows K‐selected characters with maturity at relatively large size and r‐selected characters with relatively many and small offspring, whereas ocellated puffer shows r‐selected characters with maturity at a relatively small size and K‐selected characters with relatively few and large offspring.