Reproduction related immunoglobulin changes in rainbow trout

Annual changes in plasma immunoglobulin (IgM) levels were investigated in three strains of rainbow trout Oncorhynchus mykiss which have different spawning periods, i.e., September–October, November–December, and January, reared under constant water temperature and natural day length. Plasma IgM levels decreased during the spawning season in all strains tested. The IgM changes became reversed in response to significant increases in plasma testosterone (T) and estradiol-17 in females and T and 11-ketotestosterone in males. Though the IgM decline showed a connection with suppressed immunocompetence, since many mature fish caught fungal diseases, no clear differences were observed in the plasma IgM levels between infected and noninfected fish during the spawning season. Incidentally, plasma IgM levels in infection prone fish were higher than in noninfection prone fish prior to the spawning season, whereas coincident differences in the plasma steroid levels were observed. Immature fish reared under lower water temperatures showed lower IgM levels. The effect of water temperature may have to be considered when analyzing the defense mechanism during the spawning season in rainbow trout.     

Purification,characterization and immunoassay of striped bass (Morone saxatilis) vitellogenin

The egg yolk precursor, vitellogenin (VTG), was purified from blood plasma of striped bass by chromatography on hydroxylapatite or DEAE-agarose. The fish were first implanted with estradiol-17β (E₂), which induced vitellogenesis. A rabbit antiserum (a-FSPP) raised against plasma from mature female striped bass, and then adsorbed with mature male plasma, was used to detect female-specific plasma protein (FSPP) in the chromatography fractions. Striped bass VTG (s-VTG) was collected from the peak fraction that was induced by E₂, reacted with a-FSPP, and contained all detectable phosphoprotein. It appeared as a single band (M_r ≂ 170,000) in SDS-PAGE or Western blots using a-FSPP, and as a pair of closely-spaced phospholipoprotein bands in native gradient-PAGE, suggesting that there is more than one circulating form of s-VTG. The relationship of s-VTG to the yolk proteins was verified using a-FSPP. The antiserum reacted with the main peak from gel filtration of saline ovary extracts, and it specifically immunostained the two main bands in Western blots of the extracts and the yolk granules of mature oocytes. The amino acid composition of s-VTG was similar to that of VTG from other fish and Xenopus. A radial immunodiffusion assay for s-VTG was developed using a-FSPP and purified s-VTG as standard. The s-VTG was not detected in blood plasma of males, immature females, or regressed adult females, but plasma s-VTG levels were highly correlated with plasma E₂ and testosterone levels, and oocyte growth, in maturing females. The results indicate that the maturational status of female striped bass can be identified by s-VTG immunoassay.     

Changes in plasma somatolactin levels during spawning migration of chum salmon (Oncorhynchus keta)

Plasma somatolactin (SL) concentrations were examined in chum salmon in relation to gonadal maturation; immature salmon in the Bering Sea at various stages of maturation, and mature salmon during upstream migration caught at the ocean, bay and river. Plasma SL concentrations as well as plasma prolactin (PRL) and growth hormone (GH) levels in the immature fish caught in the Bering Sea were maintained essentially at similar levels. Plasma SL in mature salmon increased significantly from the fish in the ocean to the fish in the river in both sexes. Although all the fish had fully developed gonads, females completed ovulation while still in the bay, whereas final spermeation in males was achieved after entry into the river. Thus, no clear correlation was seen between plasma SL levels and final gonadal maturation. On the other hand, plasma PRL concentrations in both male and female fish were higher in the fish in the river than those in the ocean and bay, and plasma GH levels were higher in both sexes in the fish in the bay and river than those in the ocean. Plasma levels of triglycerides, glucose, free fatty acids and ionized sodium and calcium were also examined. Significant-negative correlations were seen between plasma SL and plasma ionized calcium in mature male salmon, and between plasma SL and plasma triglycerides in mature female salmon. Although our findings do not rule out the possibility of the involvement of SL in final maturation, the results indicate that SL seems to be involved at least in energy and/or calcium metabolism during the spawning migration.     

Effects of acidic water,aluminum, and manganese on testicular steroidogenesis in <Emphasis Type="Italic">Astyanax altiparanae</Emphasis>

Metals can influence the gonadal steroidogenesis and endocrine systems of fish, thereby affecting their reproduction. The effects of aluminum and manganese in acidic water were investigated on steroidogenesis in sexually mature male Astyanax altiparanae. Whether mature male fish recover from the effects of metals in metal-free water was also assessed. The fish were exposed to 0.5 mg L^?1 of isolated or combined aluminum and manganese in acidic pH (5.5) to keep the metals bioavailable. The fish underwent 96 h of acute exposure, and samples were taken 24 and 96 h after the beginning of the experiment. The fish were then maintained in metal-free water for 96 h. Plasma levels of testosterone, 11-ketotestosterone, 17β-estradiol, and cortisol were measured. Acidic water increased the plasma concentration of testosterone and 11-ketotestosterone. Aluminum increased the testosterone levels after 96 h of exposure. Manganese increased the 17β-estradiol levels after 24 h of exposure and maintained at high levels until the end of the experiment. With the exception of acidic pH, which increased cortisol levels after 24 h of exposure, no changes were observed in this corticosteroid during the acute experiment. Aluminum and manganese together also altered steroid levels but without a standard variation. The fish recovered from the effects of most exposure conditions after 96 h in metal-free water. A. altiparanae could use reproductive tactics to trigger changes in testicular steroidogenesis by accelerating spermatogenesis and spermiogenesis, which may interfere with their reproductive dynamics.     

Effects of slow release gonadotropin releasing hormone analog on milt characteristics and plasma levels of gonadal steroids in greenback flounder, Rhombosolea tapirina

Spermiated male greenback flounder (Rhombosolea tapirina) were implanted with gonadotropin releasing hormone analog (GnRHa) pellets at different dosages to examine their effects on milt characteristics and plasma levels of testosterone (T), 11-ketotestosterone (11KT) and 17,20β-dihydroxy-4-pregnen-3-one (17,20βP). Milt and blood samples were collected for up to 28 or 42 days post-implantation (p.i.) in two separate experiments using fish that were slightly or moderately spermiated, respectively. In both experiments, fish treated with GnRHa pellets showed a consistent and significant increase in milt volume relative to controls, and the increase of milt volume was more rapid than the increase in numbers of spermatozoa. Spermatocrit (Sct) was significantly lower in GnRHa-treated fish than in controls. Plasma levels of T and 11KT were elevated at 7 and 14 days p.i. in slightly spermiated fish treated with GnRHa and elevated plasma T and 11KT levels were accompanied by increased milt volume early in the spermiation period. In contrast, there was no significant difference in plasma T levels between GnRHa-treated and control fish in fish that were moderately spermiated at the time of implant. Treatment with GnRHa tended to result in lower plasma levels of 11KT and higher levels of 17,20βP relative to controls, and there was a positive relationship between the elevation of plasma 17,20βP and the increase in milt volume in response to implantation of GnRHa pellet. Slow release GnRHa administration had no effect on sperm activity or pH and osmolality of seminal plasma in either experiment.     

光唇鱼对单色光偏好的性别差异及相关生理机制

为探究溪流性鱼类光唇鱼(Acrossocheilus fasciatus)对光色的偏好是否存在性别差异, 以及芳香化酶作为催化睾酮(T)向雌二醇(estradiol, E2)转化的限速酶在行为调节中的关键角色, 本研究采用行为学方法研究了雌鱼和雄鱼对 6 种单色 LED 光源的选择偏好, 并尝试利用药物抑制体内芳香化酶活力从而改变血浆性激素水平和鱼的行为表现。结果表明, 雌鱼和雄鱼分别相对偏好红光(峰值为 637 nm)和黄光(590 nm), 两者均相对排斥蓝光(465 nm)、 紫光(405 nm)以及含有蓝光波段的白光(447 nm), 而对绿光(518 nm)的偏好不明显。摄食含有芳香化酶抑制剂来曲唑(letrozole, LZ)的实验饲料 10 d 后, 雌性和雄性光唇鱼脑和性腺组织内芳香化酶活力和血浆 E2 水平均显著下降, 但血浆 T 水平无显著变化。值得注意的是, LZ 也导致处理鱼对光色选择偏好的改变, 雌鱼转为相对偏好蓝光, 雄鱼转为相对偏好红光。然而, 摄入含 LZ+E2 实验饲料后除芳香化酶活力降低外, T、E2 两种性激素水平和光色偏好行为表现均未出现明显变化。因此推测光唇鱼对光色偏好的性别差异极可能与体内芳香化酶活力和雌二醇水平的性别差异有关, 而与睾酮水平无明显关联。     

Changes in plasma insulin-like growth factor-I levels in the precociously maturing amago salmon, Oncorhynchus masou ishikawai

Seasonal changes in plasma levels of insulin-like growth factor I (IGF-I) in precociously maturing amago salmon (Oncorhynchus masou ishikawai), which matured as 1-year-olds, have been investigated. Profiles of plasma IGF-I levels were compared with changes in growth and maturity, and plasma growth hormone (GH) and thyroxine (T₄) concentrations. The maturity of the fish was determined by calculating the gonadosomatic index; in November, 100% of males and 89% females matured. In both males and females, plasma IGF-I levels increased from March to August, and subsequently, plasma IGF-I levels in the early maturing males and females declined gradually and were maintained at lower levels during the spawning period in November. Plasma GH levels were high in April, and then declined gradually through September. Thereafter, in early maturing fish, a slight increase in plasma GH levels was observed in October and November. No significant changes in plasma T₄ levels were found in the precociously maturing fish. In sharp contrast, plasma IGF-I levels in immature fish remained elevated through September, reaching a peak in October, and then gradually declined in November. In immature females, plasma T₄ and GH levels were elevated in August, reached their maximum in September and then gradually declined until November.     

Plasma levels of Gonadotropin-II and gonadal sex steroids in triploid catfish, Heteropneustes fossilis (Bloch)

Triploid fish have under-developed gonads due to altered reproductive endocrinology. Triploids of Indian catfish (H. fossilis) showed significantly reduced plasma levels of gonadotropin (GtH-II), testosterone (T) and estradiol-17 (E₂) than that of diploids throughout the year, except for the resting phase, irrespective of sex. Plasma levels of GtH-II were significantly different (p<0.001) between diploid and triploid fish during preparatory, prespawning and spawning phase. The plasma testosterone contents in triploids were significantly less (p<0.001) than that of diploids, except during the resting phase. Triploid females showed very low titres of estradiol-17 (<1 ng ml^–1) throughout the annual reproductive cycle in contrast to highly fluctuating levels in diploid females. Thus, this study for the first time provides information on reduced levels of GtH-II and sex steroids in plasma of male triploid fish and additional information on species-specific alteration of sex hormones in female triploids.     

The Annual Reproductive Cycle of the White Bass Morone chrysops

The gametogenic cycle of the white bass Moronc chrysops was characterized for one year at monthly intervals by measuring circulating levels of the sex steroid hormones testosterone (T) and 11-ketotestosterone (11-KT) in males, and 17β-estradiol (E₂) and T in females. Vitellogenin (VTG), the egg yolk precursor, was also measured in female blood plasma. Gonadal status of individual fish was assessed by histological evaluation, measurement of oocyte diameters, and spermintion response. At the onset of vitellogenesis (October), blood plasma levels of E₂ and T were low, but increased significantly as the spawning period (March-April) approached and oocytes attnined their maximum diameter. All stages of oocyte development were observed in mature females, indicating that the white bass is a multiple-clutch, groupsynchronous spawner. Androgen levels increased in males throughout the reproductive cycle and reached maximal levels during the spawning period. The increase in androgens coincided with sperm production and spermintion in males. The circannual endocrine and gonadal development patterns observed in white bass were similar to those of other members of genus Moronc .     

The influence of reproductive status on the stimulatory action of N-methyl-D,L-aspartate on growth hormone secretion, in vitro in rainbow trout, Oncorhynchus mykiss

The glutamate agonist, N-methyl-D,L-aspartate (NMA) stimulates the secretion of growth hormone (GH) from pituitary fragments in vitro and increases plasma GH levels in vivo in rainbow trout, Oncorhynchus mykiss (Flett et al. 1994; Holloway and Leatherland 1997a,b); however gonadal steroid hormones appear to modulate this response in experimental situations. This study examines whether steroid hormones also modulate the GH-regulatory actions of NMA during the normal reproductive cycle of rainbow trout by examining the relationship between the stage of sexual maturation and the pituitary release of GH in vitro in response to an NMA (10^-8 M) challenge. NMA had no effect on mean GH release from the pituitary glands of fish that were immature (GSI <1.0), from males during early development (GSI 1.0-3.0), or from sexually mature males (with free running milt) and females (ovulated). However, NMA significantly increased GH release from pituitary glands taken from females during the early stages of gonadal growth (GSI 1.0-9.0) and from males and females sampled during the later stages of gonadal growth (males GSI 3.01-6.0; females GSI 9.01-15.0). The GH-stimulatory action of NMA in males and females progressed to a maximum effect during the late stages of gonadal growth, and disappeared in ovulated females and free running males. Moreover, in female fish, the maximal GH release in response to the NMA challenge is positively correlated with plasma 17β-estradiol levels; no such correlation was evident for plasma testosterone levels in males. Changes in the GH response to NMA during maturation while gonadal steroid levels fluctuate provides further evidence to suggest that the effects of NMA on GH secretion are intimately linked to endogenous gonadal steroid hormone levels. This revised version was published online in July 2006 with corrections to the Cover Date.     

Seasonal reproductive cycle of Waigieu seaperch (Psammoperca waigiensis)

Predictive and reliable parameters of reproductive status are integral aspects of sustainable fisheries and aquaculture management. These parameters are also important for an accurate evaluation of the effects of different treatments on sexual maturation in fish farming. In the present study, we have characterized the seasonal reproduction profile and described changes in sex steroids in relation to gonadal maturation and development in female and male Waigieu seaperch (Psammoperca waigiensis). The experimental period covered a full calendar year (January–December). In males and females, we observed that plasma sex steroid hormones [oestradiol‐17β (E2), testosterone (T), 11‐ketotestosterone (11‐KT) and progesterone (P)] levels showed monthly fluctuations during the spawning period. Particularly, plasma steroid hormone levels were positively associated with gonadosomatic index values. In addition, high levels of plasma steroid coincided with recruitment of oocytes into yolk accumulation in females. The main spawning period occurred between April and October in females, and between March and November in males. The non‐aromatizable androgen, 11‐KT is generally believed to be the active male‐specific androgen in teleosts, and is associated with the process of spermiation, development of secondary sexual characteristics and regulation of male reproductive behaviour in most teleost species. In this study, we found relatively high amounts of 11‐KT in females between May and December, suggesting an integral role in the maturation process, also for the females. A rapid peak in plasma P level was observed in November and suggests significant roles during post‐spawning and/or resting periods in both female and male fish. Furthermore, all oocyte developmental stages were present within the same sampling month and also within the spawning period, demonstrating the gamete group asynchronous developmental strategy. Overall, Waigieu seaperch showed strong seasonality in reproductive development with corresponding sex steroid patterns. The data presented in this study may contribute to the understanding of the reproductive endocrinology of a tropical marine finfish with increasing industrial prospects and sustainable aquaculture of this species in a developing country, such as Vietnam.     

Effects of hemi-castration on plasma steroid levels in two teleost fishes; the three-spined stickleback, Gasterosteus aculeatus, and the Atlantic salmon, Salmo salar

In order to study the possible homeostatic regulation of gonadal steroids in fishes, plasma steroid levels were measured in hemi-castrated and sham-operated nesting male three-spined sticklebacks, Gasterosteus aculeatus, and in mature 2-year old male Atlantic salmon, Salmo salar. Hemi-castration significantly suppressed androgen levels in both species. In sticklebacks, plasma levels of 11-ketotestosterone (11KT) were 56% and levels of testosterone (T) 55% of those found in sham-operated males. In hemi-castrated salmon the levels of 11KT were 63%, and the levels of T were 75% of the levels in sham-operated males. In contrast, levels of 17α,20β-dihydroxy-4-pregnen-3-one (17,20-P) in salmon (not measured in sticklebacks) were not different between hemi-castrated and sham-operated males. The results suggest that, although levels of the steroid 17,20-P were compensated in hemi-castrated salmon, the androgen levels in fish males in full spawning condition are not closely regulated by negative feedbacks. This revised version was published online in July 2006 with corrections to the Cover Date.     

Effects of Dietary Protein Levels on Gonad Maturation, Size and Age at First Maturity, Fecundity and Growth of Nile Tilapia

Six-week-old Nile tilapia (Oreochromis niloticus) fry with an average weight (SD) of 0.51(0.2) g were reared for 140 days on five formulated, isocaloric diets of different protein levels (25, 30, 40 and 45% by dry weight). Fish fed diets of higher protein levels (40 and 45%) showed better growth and feed conversion ratio than those on lower protein levels. Fast-growing fish matured earlier. Maturation rate was affected by the dietary protein levels. Males matured earlier than females: the first mature males were recorded when they were 14 weeks old, whereas the females matured after 18 weeks. In both sexes, mean percentage of mature fish rose with increasing dietary protein level, the percentage of mature males being higher than that of the females. Similarly, the percentage of mature fish rose with the increasing age of fish, with more than 50% males and females mature at the age of 22 and 24 weeks, respectively, the exception being the 25% protein diet fed fish, where the percentage of mature fish was below 50%. In all treatments, spawning was initiated when the fish were 22 weeks old. Smallest size at spawning of males and females was 9.2 cm (13.1 g) and 8.1 cm (8.9 g), respectively, and dietary protein levels influenced the size of fish at first maturity. For both sexes, no difference was found in the gonado-somatic index (GSI) among different treatments. Fecundity increased with increasing dietary protein levels, but significant differences were found only between 40–45% and 25–35% dietary protein levels. The relative fecundity (eggs g^–1 female) was higher at the lower dietary protein levels (25–35%) than at the higher dietary protein levels (40–45%). The dietary protein levels did not have any significant influence on the size and weight of mature eggs. The chemical composition of fish and mature ovaries was significantly influenced by the dietary protein level.     

Effects of hormonal manipulation on stress responses in male and female broodstocks of pikeperch Sander lucioperca

This study was designed to determine the effects of hormonal manipulation on stress responses in female and male pikeperch. Two-year-old cultured female and male broodstocks with an average weight of 337.4 ± 20.1 (mean ± SE; n = 16) and 318.7 ± 15.1 g (n = 16), respectively, were randomly allocated into four hormonal treatments each containing 4 fish. Two sexual groups of 16 fish for each gender were considered. Sexually mature male and female pikeperch were injected with either physiological saline solution (as control group), common carp pituitary extract (CPE), human chorionic gonadotropin (hCG) and or luteinizing hormone-releasing hormone analog (LHRHa₂). The blood samples were taken before hormonal injection and after ovulation and spermiation. Then the plasma levels of stress indices (cortisol, glucose, and lactate) were determined. The results showed that all CPE-, HCG-, and LHRHa_2- injected males produced sperm. In females treated with CPE and hCG, three of four ovulated, but none of LHRHa_2- and saline-injected fish spawned. Significant changes in cortisol, glucose, and lactate levels were observed among the females injected with different hormones. Plasma cortisol and glucose levels increased significantly in males injected with CPE and females injected with hCG, but no significant change was observed in lactate levels before and after hormonal induction. Comparison of two sexes revealed significant differences in glucose levels for females in some groups before injection, while CPE-injected sexes showed significant changes in cortisol and lactate concentrations. The results indicated that the induction of ovulation or spermiation stimulated stress responses especially in female pikeperch, and therefore, all the procedures should be made to minimize the disturbance during the artificial spawning.     

Disparate effects of gonadal steroid hormones on plasma and liver mRNA levels of insulin-like growth factor-I and vitellogenin in the tilapia, Oreochromis mossambicus

The tilapia, Oreochromis mossambicus, exhibits a sexually dimorphic pattern of growth, males growing larger than females. We examined the effects of E₂ and DHT on the GH/IGF-I axis and on VTG production in the tilapia. Sexually mature tilapia were injected with 5 μg g body weight of E₂ (males) or DHT (females) every 5 days for a total of 3 injections. Female tilapia had significantly higher plasma GH levels than males. However, plasma and liver mRNA levels of IGF-I were significantly lower in females than in males, whereas VTG levels in both the plasma and liver mRNA were significantly higher in females than in males. Although significant amounts of VTG were detected in control males (8 ± 0.3 μg ml), the levels in control females (3000 ± 500 μg ml) were about 400 times higher than in males. Males treated with E₂ exhibited a female-like GH/IGF-I profile. That is, they had significantly elevated levels of plasma GH with lower plasma IGF-I and liver IGF-I mRNA levels. Estradiol treatment significantly elevated both plasma and liver mRNA VTG levels. Dihydrotestosterone treatment in females induced a male-like GH/IGF-I profile: plasma GH levels were significantly reduced, whereas plasma and liver IGF-I mRNA levels were significantly elevated. Both plasma and liver mRNA levels of VTG were not altered by DHT treatment. Pituitary GH mRNA levels were similar in all treatment groups. These results clearly indicate that estrogens and androgens feminize and masculinize the GH/IGF-I axis, respectively. This revised version was published online in July 2006 with corrections to the Cover Date.     

Determination of maturity in male hawksbill turtle <Emphasis Type="Italic">Eretmochelys imbricata</Emphasis> in captivity based on tail elongation and plasma testosterone level

To determine the sexual maturity of fourteen male wild-caught hawksbill turtles [straight carapace length (SCL) range 63–79 cm] held in captivity, we investigated their hormone levels in blood and morphological characteristics. Male turtles were divided into two groups: five individuals showing mating behavior (group A) and nine individuals not showing mating behavior (group B). Then, seasonal changes of plasma testosterone of both groups were studied. We also tried to determine male maturity based on tail elongation (TE) index, i.e., the ratio of tail length (TL) to SCL. Plasma testosterone level of group A gradually increased during premating and then sharply declined to low levels between mating and postmating seasons. In contrast, that of group B remained low during the experimental periods. The distinct seasonal changes of plasma testosterone levels of groups A and B correlated with their maturity. Furthermore, males were clearly divided into mature and immature based on TE values >0.35 and <0.33, respectively, corresponding to results based on plasma testosterone levels. Our results indicate that TE can be used more conveniently and accurately to determine maturity of male hawksbill turtles in captivity.     

Plasma vitellogenin levels in male common carp Cyprinus carpio and crucian carp Carassius cuvieri of Lake Kasumigaura

In order to investigate the possible influence of estrogenic environmental endocrine disruptors on the reproductive activity of fish in Lake Kasumigaura, plasma levels of vitellogenin (VTG), a biomarker of estrogen exposure, were measured in wild and cultured male common carp Cyprinus carpio and wild crucian carp Carassius cuvieri . Testicular histology and plasma steroid hormone levels were also examined. Fish were collected from June 1998 to August 1999. Plasma VTG levels in most fish examined were below a detection limit (40 ng/mL) throughout the sampling period, and a small amount of VTG (43.5 ng/mL–1680 ng/mL) compared to that in females was detected in some fish. Active spermatogenesis in the testis and increased levels of plasma sex steroids were observed in most of the fish examined. Thus, no marked influence of estrogenic chemicals was detected in the reproductive activity of male common carp and crucian carp in Lake Kasumigaura collected from the sampling sites in the present investigation.     

Sexual patterns and protogynous sex reversal in the rusty parrotfish, Scarus ferrugineus (Scaridae): histological and physiological studies

Gonadal histology confirmed that Scarus ferrugineus is a diandric protogynous fish. The process of protogynous sex reversal was investigated through histological observations on the gonads of females changing sex to male. This process was divided into three stages on the basis of changes in the structure of the germinal and somatic elements. Ovaries of functional females (stages IV-V) were filled with vitellogenic oocytes during the breeding season but contained no trace of spermatogenic tissue. During post-spawning period, the remaining vitellogenic oocytes began to degenerate and accompanied by a drop in plasma levels of estradiol-17β. At the commencement of sex change, previtellogenic oocytes began to degenerate and stromal cell aggregation was observed in the central region of the lamellae. At mid-reversal stage, steroid-producing cells (Leydig cells) developed at the border of the stromal aggregate and spermatogonial cysts appear at the periphery of lamellae. Finally, sex change to secondary males was considered complete, with the beginning of active spermatogenesis and spermiation. Plasma levels of testosterone remained low throughout the sex change, but II-KT increased rapidly parallel to the increased number of Leydig cells while the level of estradiol-17β decreased. The results indicate also that the sex-changed males had higher level of II-KT than primary males, while primary males had higher level of testosterone. Histological examination revealed that testes of primary and secondary males are almost identical in organization of the spermatogenic cysts, association of sertoli cells, and developing germ cells but differ in clustering and development of Leydig cells.