Effects of transplants and extracts of thoracic nerve cord–ganglia on gonad maturation of penaeoid shrimp

It has been established recently that interspecific and intraspecific thoracic ganglia transplants from Penaeidae are gradually absorbed by the host without activating an encapsulation mechanism. Therefore, this research was designed to evaluate the thoracic ganglia extracts and implants from maturing Trachypenaeus byrdi (Burkenroad), Xiphopenaeus riveti (Bouvier) and Penaeus (Litopenaeus) occidentalis (Streets) females as potential inducers of sexual maturation in Penaeus ( Litopenaeus) stylirostris (Stimpson), Penaeus (Litopenaeus) vannamei (Boone) and T. byrdi , from the Gulf of Nicoya, Costa Rica. Our findings suggest that interspecific and intraspecific thoracic ganglia extracts or implants from maturing penaeoid females are not capable of inducing a clear response in sexual maturation in males or females. Tissues were tested at increasing doses from 137, 386, 525 and 1500 μg g⁻¹ body weight, without any positive response. It is proposed that a hypothetical hormone, vitellogenesis-stimulating hormone, from the thoracic ganglia, is under the strong negative control of eyestalks, by the gonad-inhibiting hormone in the subgenus Litopenaeus . Therefore, the use of thoracic ganglia extracts or implants would be ineffective when compared with injecting serotonin alone, as the present results seem to support.     

Oocyte Growth in the Striped Mullet Mugil cephalus L. Maturing at Different Salinities

Profiles of oocyte growth were obtained from female striped mullet Mugil cephalus L. held in salinities ranging from fresh water to seawater during two consecutive spawning seasons (1988–1990) in Hawaii. Females underwent vitellogenesis at all salinities (i.e., 32–35, 13–20, and 0%‰) tested. Females maturing in fresh water exhibited a slower rate of oocyte growth, and a significantly lower number completed vitellogenesis. All females were induced to spawn in full seawater. The number of fertilized eggs per spawning was highest from females maturing in brackish water. More females were able to be induced to spawn twice in brackish water during the 1989–1990 season than in fresh or seawater. The results from the current investigation suggest that salinities ranging from 13–35 ppt are adequate for ovarian maturation in captive striped mullet females.     

Ovarian maturation stages of the mud crab Scylla serrata

Ovarian maturation in adult wild‐sourced and pond‐grown Scylla serrata (Forsskål) was determined based on gross morphology and histological appearance. There were no significant differences noted in the histological features of both wild and pond‐reared S. serrata females. Ovarian maturation was classified into five stages: immature, early maturing, late maturing, fully mature and spent. The immature ovaries are thin and translucent to off white and contain oogonia, primary oocytes with large nuclei. The follicle cells were found around the periphery of the lobes and an area among groups of oogonia and oocytes. The follicle cells gradually enclosed the oocytes. The early‐maturing ovaries were yellow and small yolk globules started to appear in larger oocytes. In late‐maturing ovaries, the colour became light orange and lobules were apparent. Yolk globules occurred in the cytoplasm with larger globular inclusions towards the periphery, while follicle cells were hardly recognizable. Fully mature ovaries were orange to deep orange and had swollen lobules. Large yolk globules were apparent in the entire cytoplasm. Follicle cells were hardly seen. Spent ovaries were similar to the early‐maturing and late‐maturing stage in partially spawned females. The ovarian development was correlated closely to the gonadosomatic index, oocyte diameter, and ovarian histology. The classification of ovarian maturation provides baseline information for further studies on reproductive biology. Likewise, the information provides a guide for broodstock management in the hatchery.     

In vitro study of a putative role of gonad‐inhibiting hormone in oocyte growth stimulation in Penaeus monodon

Ovarian development in crustacean is controlled by several factors, among which a neuropeptide gonad‐inhibiting hormone (GIH) is known to inhibit vitellogenin (Vg) synthesis in the ovary. It has been postulated that GIH may control Vg synthesis by inhibiting the release of gonad‐stimulating factor (GSF) from brain and thoracic ganglia. To prove this hypothesis, this study was primarily aimed to investigate the influence of GIH on the release of GSF from thoracic ganglia of Penaeus monodon. Our result showed that GIH did not suppress the release of putative GSF from thoracic ganglia by calcium ionophore A23187 as the induction of oocyte growth in the ovary explants that were cocultured with thoracic ganglia in the presence of A23187 was not affected by the addition of recombinant GIH protein. In addition and interestingly, when the ovary explants were incubated with the recombinant GIH alone, the oocyte growth was increased at the rate comparable to that induced by A23187 in the presence of thoracic ganglia. Hence, our in vitro study demonstrated that the stimulation of GSF released from thoracic ganglia is independent of GIH, and that the GIH has a dual function in oocyte growth stimulation and inhibition of Vg synthesis in the early stage of ovarian development. This expands our knowledge on the regulation of ovarian development in shrimp by GIH. Further in vivo studies in this novel aspect of GIH function will be useful for the improvement of shrimp ovarian maturation in the future.     

Induced Ovulation of Southern Flounder Paralichthys lethostigma Using Gonadotropin Releasing Hormone Analogue Implants

Implanted pellets that provide a sustained release of [D-Ala⁶ Des-Gly¹⁰] LHRH-ethylamide (GnRHa) were used to induce maturation and ovulation of Southern flounder Paralichthys lethostigma . Of the 12 females whose ovaries contained follicles with a maximum diameter ≥500 μm, 11 ovulated for the first time within 90 h of hormone implantation. Only 1 fish with a maximum follicle diameter less than 500 μm ovulated within 2 wk after implantation. Ovulated eggs were manually stripped from the females and mixed with sperm from several males. Most females were spawned 1 to 3 times on consecutive days with variable fertility. One female was spawned 11 times producing 668,000 eggs. Fertility was evaluated by examining the incubated eggs for early stages of embryonic cleavage. The percentage of fertile eggs in subsamples of incubated eggs ranged from 7–95%. The results indicate that GnRHa implants can be used to induce repeated ovulation in this species. The variability in fertility is discussed in relation to egg quality.     

Induced maturation and spawning: opportunities and applications for research on oogenesis

Broodstock management requires the ability to detect and regulate oocyte growth, acquisition of maturational competence, maturation of oocytes, and onset of ovarian atresia. Our research on temperate basses (genus Morone) has supported development of these capabilities. These investigations have revealed that accumulation of neutral lipid droplets and deposition of vitellogenin-derived yolk proteins in growing oocytes are independent processes with different sensitivities to changing day length and water temperature. In these fishes, completion of oocyte growth is marked by disappearance of vitellogenin from ovarian biopsy samples. Competence of females for induced spawning is predicted by the ability of biopsied follicles to initiate oocyte meiosis in vitro in response to insulin-like growth factor I. Cytoplasmic maturation of the oocytes is triggered by the maturation-inducing steroid hormone and can be monitored by evaluating degradation of the yolk proteins. Onset of ovarian atresia is indicated by the appearance of edema in the granulosa cell layer of biopsied follicles, and can be delayed for months by holding gravid females at abnormally low temperature (`cold banking'). These novel findings hold strong promise for application to other farmed fishes.     

锯缘青蟹促雄腺和胸腹神经团的直接联系

采用组织学、扫描电镜方法对锯缘青蟹(Scylla serrata)促雄腺和胸腹神经团之间的关系进行观察。结果表明，射精管末端存在促雄腺，促雄腺和胸神经团来源的支配第五步足(即游泳足)的神经紧密相连。该神经在游泳足基部的肌肉内部紧贴射精管并行，其分支支配着射精管周围肌肉，可能也是控制精荚排放的神经。本研究可为促雄腺对性别控制的机制及蟹类精荚电促排的深入研究提供理论依据。     

Tissue Lipid Content and Fatty Acid Composition of Penaeus monodon Fabricius Broodstock from the Wild

Tissues (hepatopancreas, muscle, gonad) of wild Penacus mordon females with ovaries in different stages of development and males were analyzed for Upid content and fatty acid composition. Hepatopancreatic tissue had the highest mean levels of lipid at 15.72–25.20% in females and averaged 46.20 ± 1.53% in males. Levels of Lipid in the muscle were low with no marked variation throughout the maturation s-es. Ovarian lipid levels increased significantly at stage II (early maturing ovaries) and remained high through stage IV (fully mature ovaries). Peak lipid level was found at stage III (late maturing ovaries) and coincided with a decline in hepatopancreas lipid. Ovarian and hepatopancreatic lipids declined drastically in spent females. Profiles of fatty acid in the tissues consistently showed the presence of PUFA 204n-6, 20:5n-3 and 22611-3, with high levels in the polar lipid fraction of vitellogenic ovaries. These values were reflected in the male testis and spawned eggs. The finding suggest storage and utilization of lipids for maturation and spawning processes and that the phospholipids were mainly responsible for increases in ovarian lipids. The type of fatty acids present in maturing prawn ovaries is indicative of their importance in reproductive physiology.     

The growth and reproductive endocrinology of adult triploid Pacific salmonids

This paper describes the effect of triploidy on growth and reproductive endocrinology in the months leading up to and including spawning in rainbow trout,Salmo gairdneri, and pink salmon,Oncorhynchus gorbuscha. Growth rates were the same for diploid and triploid rainbow trout, but triploid female pink salmon were smaller than maturing diploid females and diploid and triploid males of the same age. Triploid males of both species developed typical secondary sexual characteristics and had normal endocrine profiles, although their cycle appeared to be delayed by about one month. Triploid females remained silvery in appearance and showed no endocrine signs of maturation, even at the level of the pituitary. Thus, although triploids of both sexes are genetically sterile, only the females do not undergo physiological maturation.Reported in part at the Third International Symposium on Reproductive Physiology of Fish, St. John's, Newfoundland, August 2–7, 1987.     

Changes in alkaline phosphatase isoenzyme activity in tissues and plasma of Atlantic salmon (Salmo salar) before and during smoltification and gonadal maturation

Changes in tissue and plasma isoenzymes of alkaline phosphatase (ALP) were qualitatively and quantitatively determined for male and female Atlantic salmon parr, silvery parr, smolt, immature grilse, prespawning grilse and postspawning grilse using cellulose acetate electrophoresis, densitometry and spectrophotometry. Tissue ALP isoenzymes were isolated from intestine, kidney, bone, liver, and gonad and compared to plasma isoenzymes. Parr plasma displayed three isoenzymes from bone and liver (slow and fast). During smoltification, ALP activity increased in tissue extracts from liver, gonad, and kidney of males and females. Total plasma ALP activity also increased and was due to slow and fast liver isoenzymes. During ovarian development, total ALP plasma activity increased in females and was due mostly to liver isoenzymes and an incompletely identified isoenzyme or isoenzyme mixture (band 2). However, in males total ALP plasma activity did not increase during maturation and no band 2 was evident. In male and female maturing adult grilse, bone ALP activity declined and the isoenzyme band evident in parr plasma could not be detected. ALP activity declined in the plasma of postspawning males and females. In females this was due partly to the total clearance of band 2 from the plasma, together with lowered levels of liver isoenzymes. Treatment of postspawned grilse in February and March with triiodothyronine and thyroxine elevated plasma thyroid hormone levels and increased plasma ALP levels. In conclusion, plasma ALP isoenzyme activities change with physiological state, and knowledge of the conditions governing these changes is important when using these enzymes as a diagnostic tool.     

Effects of insulin-like growth factor-I on final oocyte maturation and steroid production in Fundulus heteroclitus

Recent evidence has indicated the presence of IGF-I and IGF-I receptors in mammalian and teleost ovarian follicles. Since growth hormone (GH), which can be secreted from the pituitary concomitant with a gonadotropin as a response to gonadotropin-releasing hormone, generally acts to release IGF-I from tissues including the ovary, the effect of IGF-I itself on ovarian steroidogenesis and oocyte maturation was investigated in the model teleost, Fundulus heteroclitus. IGF-I was found to be without effect on ovarian follicle steroidogenesis, but initiated oocyte maturation in a dose-dependent manner even more rapidly and effectively than 17,20-dihydroxy-4-pregnene-3-one (DHP), the naturally occurring maturation-inducing steroid. IGF-II also induced oocyte maturation in a dose-dependent manner. IGF-I induction of oocyte maturation occurred in the absence of DHP production by the granulosa cells (which is normally stimulated by gonadotropin), and could be inhibited by cycloheximide but not actinomycin D, thus implicating the role of protein synthesis. These results suggest that GH-stimulated release of ovarian IGF-I may have an even more direct role than DHP on the reinitiation of oocyte maturation.     

三疣梭子蟹卵巢发育过程中雌二醇的免疫定位和变化

雌二醇(estradiol,E_2)是甲壳动物体内重要的性类固醇激素,对其卵巢发育和卵黄发生起着十分重要的调控作用。本实验采用免疫组织化学方法系统研究了三疣梭子蟹(Portunus trituberculatus)卵巢发育过程中E_2在卵巢、肝胰腺、胸神经节、脑神经节和大颚器中的分布及变化。结果表明:(1)蟹卵巢中E_2的免疫阳性主要分布于滤泡细胞和卵巢发育中后期的卵母细胞细胞质(II-V期),卵巢发育过程中卵母细胞细胞质和细胞核中的E_2免疫阳性均为先上升后下降的趋势,最大免疫阳性分别出现在IV期和II-III期,滤泡细胞中始终存在较强的E_2免疫阳性;(2)肝胰腺中的E_2免疫阳性主要分布于F细胞及R细胞细胞核中,R细胞细胞核中的E_2免疫阳性从III期开始显著下降,F细胞中始终存在较强的E_2免疫阳性;(3)卵巢发育(I-V期)的神经组织,E_2主要分布于胸神经团的神经细胞和神经髓质以及脑神经节神经细胞细胞核内,其中脑神经节神经细胞细胞核中始终保持着强免疫阳性,卵巢发育早期(I-II期)胸神经团中的神经髓质中为E_2中等免疫阳性,神经细胞中一直为弱免疫阳性;(4)就大颚器而言,E_2强免疫阳性始终存在于大颚器腺细胞的核仁以及细胞核周围的细胞质内。以上结果表明,E_2在三疣梭子蟹卵巢、肝胰腺、神经组织和大颚器中广泛分布,且免疫阳性与卵巢发育具有一定的相关性,E_2可能通过作用于多个靶器官来调控三疣梭子蟹的卵巢发育。     

The effect of whole body lipid on early sexual maturation of 1+ age male chinook salmon (Oncorhynchus tshawytscha)

Early sexual maturation of male chinook salmon (maturation 1 to 4 years prior to females in the same age class) results in reduced effectiveness of stock enhancement programs and a financial loss to the salmon farming industry. Previous studies in Atlantic salmon have shown that the age of maturity in males is affected by growth and/or body energy stores, but the relative roles of these two factors are not well understood. Therefore, an experiment was designed to determine when spermatogenesis was initiated, to characterize the endocrine changes during the onset of puberty in male salmon, and to determine if the level of whole-body lipid affects the incidence of early male maturation in a wild stock (Yakima River) of 1+ spring chinook salmon. Fry were fed a commercial diet from February until August and were then divided into groups of 320 fish (mean weight, 5.6 g) and fed one of five experimental diets (two replicate groups/diet) containing 4%, 9%, 14%, 18% or 22% lipid and 82%, 77%, 73%, 69%, or 65% protein for 13 months. Fish were reared on natural photoperiod and ambient temperature (6°C to 16°C), and pair-fed to a level based on the tank with the lowest feed consumption. Fish were weighed monthly and sampled to determine body composition, pituitary follicle-stimulating hormone (FSH) and luteinizing hormone (LH) levels, plasma insulin-like growth factor I (IGF-I) levels, and stage of gonadal development.

Throughout the experimental period the mean fish weight was similar among treatment groups. However, from December through the end of the experiment in the following September, maturing males were significantly larger than nonmaturing fish. Initial lipid levels in 0-age experimental fish were near 6%, which is similar to wild fish of the same stock and age captured in the Yakima River during August. Fish fed diets containing more than 4% lipid increased in whole-body lipid content during the first 2 months of feeding and then maintained at relatively constant levels during the course of the experiment. Whole-body lipid levels for the dietary treatment groups averaged 5.6%, 7.1%, 8.2%, 9.4%, and 9.6% from October through the following September.

Based on histological examination of the testes of experimental fish, type B spermatogonia and primary spermatocytes were first observed in some of the yearling males during November. These were designated maturing males. Pituitary FSH levels were significantly higher in maturing than nonmaturing males at this time and for the remainder of the study. Pituitary FSH levels increased as spermatogenesis proceeded in maturing fish, whereas pituitary LH levels increased in maturing 1+ males only during July and August, when testes were in late stages of spermatogenesis and in September during spermiation. Plasma IGF-I levels were significantly higher in maturing males than nonmaturing fish from December through the end of experiment. Since maturing males were significantly larger than nonmaturing fish of both sexes from December through September, the difference in IGF-I levels could be due to differences in growth or due to maturation.

The percentage of maturing males was significantly influenced by whole-body lipid, increasing from 34% in fish fed the 4% lipid diet to 45% in fish fed the 22% lipid diet. These data suggest that whole-body lipid levels influenced the incidence of maturation of male spring chinook salmon. In addition, both endocrine and histological indicators suggest that maturation was initiated in males approximately a full year prior to the time the fish will spawn.     

Induction of spawning in Pacific white shrimp Litopenaeus vannamei (Boone, 1931) by injection of its molt inhibiting hormone isoform II produced in E. coli

The optimization of reproductive parameters in shrimp farming continues to be a challenge for most producing countries. Although the crustacean neuropeptides have been studied extensively in the last two decades, the functions of most of these neuropeptides remained putative. Among them, molt‐inhibiting hormone isoform II (MIH II) has shown an important role in vitellogenesis. In this study, the cDNA encoding mature MIH II peptide was isolated by RT‐PCR from the L. vannamei eyestalk. The cDNA was cloned into pET28a bacterial expression vector. Recombinant MIH II was obtained in the form of insoluble inclusion bodies and purified to ~88% purity. Two doses of rMIH II and a negative control group were assayed in vivo. The stages of ovarian maturation and spawning were recorded during 72 hr post‐injection. The results showed that ovarian maturation occurred approximately in 9% and 33% of females injected with rMIH II at the doses of 300 and 600 ng/gbw respectively. Neither maturation nor spawning was detected in the negative control group. Females injected with 600 ng/gbw, which showed vitellogenic stages III and IV, spawned. These preliminary results argue that the hormone rMIH II could be a promising candidate to induce spawning in L. vannamei shrimp.     

Lipids and fatty acids in wild and pond-reared mud crab Scylla serrata (Forsskål) during ovarian maturation and spawning

Wild‐caught and pond‐reared female mud crab Scylla serrata at different stages of ovarian maturation were collected from Samar and Capiz, Philippines. Crabs were categorized into five stages according to the external morphological and microscopic appearance of the most advanced oocytes. The ovaries, hepatopancreas, muscle and newly spawned eggs (NSE) were analysed for lipid class components and fatty acids. Total lipid was higher in pond‐reared than in wild‐caught crabs but increased with ovarian maturation in both groups. Ovarian lipid peaked at the fully mature stage, coinciding with a decline in hepatopancreatic and muscle lipids. Lipid levels declined significantly in spent females. The tissues contained elevated highly unsaturated fatty acids such as arachidonic (20:4n‐6), eicosapentaenoic (20:5n‐3) and docosahexaenoic (22:6n‐3) acids, but at higher levels in late maturing and fully mature ovaries and in NSE. The type of lipid class and fatty acid components in mature ovaries as well as in NSE are generally considered to be indicative of their importance in reproductive physiology and embryonic and larval development.     

中华绒螯蟹离体卵巢发育的调控作用

通过卵巢离体实验研究性激素、大豆黄酮、生物胺及神经组织对中华绒螯蟹(Eriocheir sinensis)卵巢发育的影响。结果表明，雌二醇可显著地增大卵黄发生前期、初级卵黄发生期和次级卵黄发生期卵巢卵母细胞的直径，黄体酮可显著增大卵黄发生前期卵巢卵母细胞直径，对初级卵黄发生期和次级卵黄发生期的卵母细胞直径有极显著地促进作用；大豆黄酮具有增大卵母细胞直径的作用，但作用不显著；脑和胸神经团对卵黄发生前期、初级卵黄发生期的卵母细胞直径有显著增大作用，对于次级卵黄发生期卵母细胞直径有极显著增大作用；视神经节作用不明显；5-羟色胺通过脑可显著促进卵黄发生前期、初级卵黄发生期卵巢卵母细胞直径的增大，极显著促进次级卵黄发生期卵巢卵母细胞直径的增大，还通过胸神经节极显著增大各期卵巢的卵母细胞直径；多巴胺作用不明显。     

Impact of long‐term dietary exposure to lead on some reproductive parameters of a female Common carp (Cyprinus carpio L.)

During a long‐term 3‐year dietary exposure of mature carp females to lead, its bioaccumulation in the brain, changes in the neurohormonal activity at the level of the hypothalamus and the pituitary gland as well as the growth and maturation of ovaries were analysed. Moreover, an analysis of the effectiveness of hormone‐stimulated spawning during two subsequent spawning seasons was carried out. The results of the analyses show that chronic exposure of maturing and mature carp females to lead in feed results in its accumulation in the brain (1.365 μg g^?1). This impairs the endocrine activity of the hypothalamus, which is manifested by, among others, an increased secretion of dopamine and impaired spontaneous secretion of luteinizing hormone (LH). The observed decrease in the concentration of gonadotropin in blood inhibits vitellogenesis, which is manifested by a lower degree of the maturity of oocytes, lower fecundity (as measured by egg number, egg weight, egg maturity and egg survival) and lower gonadosomatic index values. The final negative impact of lead is the impairment of reproductive functions, as manifested by a smaller number of spawning females and in their lower fecundity during spawning.     

Effects of melatonin implants on reproduction and growth of turbot broodstock

The effect of 18 mg. melatonin (MLT) implants on thereproductive performance of turbot (Scophthalmus maximusL.) broodstock was determined in 20 females and 12 males. Implantedanimals showed supra-physiological plasma MLT concentrations during threemonthsafter implantation, being at that time about one hundred times higher thandiurnal MLT concentrations. Direct effects of MLT implants were observed ongonadal maturation of turbot females three months after implantation (P <0.01), although the faster ovarian response to increasing photoperiod was notconsistent and no differences were detected at the final reproductiveperformance. MLT implants had a positive effect on turbot male reproductivefeatures, since gonadal recrudescence started 45 days earlier (P < 0.05)showing a trend towards higher spermatozoa concentration. Further research isadvisable to ascertain the MLT dose required for and time of implantation inturbot broodstock.