Injections Of Salmon Pituitary Extract Research Articles

Development and validation of enzyme-linked immunosorbent assays specific for follicle-stimulating hormone and luteinizing hormone in Japanese eel

Two types of gonadotropins (Gths), follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh), are primary regulators of reproduction in vertebrates, including teleost fish. In this study, recombinant Japanese eel Gths (reGth; reFsh and reLh), and recombinant chimeric Gths (rcGth; rcFsh and rcLh), consisting of an eel β-subunit with rabbit glycoprotein hormone α-subunit, were produced as single-chain proteins, using a mammalian expression system and subsequently highly purified. Antibodies raised against rcGths were affinity purified with the corresponding reGth, and specific enzyme-linked immunosorbent assays (ELISAs) for Japanese eel Fsh and Lh were developed using these antibodies for the first time. The ELISAs were sensitive and parallelism was seen between the standard curve and serial dilutions of Japanese eel serum and pituitary extract (PE). Contents of Fsh and Lh protein in the pituitaries of the female Japanese eel at the pre-vitellogenic stage were comparable and the Lh contents at the migratory nucleus stage were highly induced during artificial induction of maturation by injections of salmon PE. Furthermore, the accumulated Lh was released at a high level by administration of a Lh-releasing hormone analogue and pimozide. Recombinant Gths and homologous ELISAs established for Japanese eel may be useful tools for studying reproductive eel biology.

The Relationship between the Developmental Stage of Oocytes in Various Seasons and the Quality of the Egg Obtained by Artificial Maturation in the Feminized Japanese Eel Anguilla japonica

Feminized 2-year-old eels were reared in freshwater at 26℃ until October, then the water temperature was gradually decreased to 16℃ to December, and gradually increased to 26℃ from February to April. They received weekly injections of salmon pituitary extract (body weight, 15 or 30 mg/kg) to induce sexual maturation in September, December, or April. The quality of the eggs obtained in each season was evaluated by the resulting fertilization, hatching, and 8-day sur- vival rates. High quality eggs were obtained via high-dose injections in most experimental groups. In the high-dose injection groups, eels were induced to the final maturation phase in all seasons; however, egg quality obtained in April was extremely low in contrast to that obtained in September and December. Histological observation of oocytes just before the process of artificial maturation was commenced indicated that the developmental stages of oocytes varied by season: oil droplet in September, early vitellogenic in December, and regressive from vitellogenesis in April. The extremely low egg quality in April is probably due to the fact that the oocytes had already entered the regressive stage. These results indicated that better quality of eel eggs can be obtained by starting the process of artificial maturation between September and December.

Fusion of lipid droplets in Japanese eel oocytes: Stage classification and its use as a biomarker for induction of final oocyte maturation and ovulation

The quality of eggs obtained from maturation-induced Japanese eel, Anguilla japonica, is unstable. One of the causes for low quality eggs is that females are artificially induced to ovulate at an inappropriate maturational status. Here we investigated the feasibility to use the morphological changes of lipid droplets in oocytes as a biomarker to show the optimum timing for injections of salmon pituitary extract (SPE) for priming and maturation-inducing steroid (MIS) for final oocyte maturation and ovulation. Various sizes of oocytes (700 to 1000 μm) were collected by cannulation from maturation-induced female eels during five days until ovulation. Morphology of the lipid droplets in these oocytes were classified into 10 stages mainly on the basis of their diameter, which increased with the progress of maturation as the droplets fused together. To assess the maturational status of each female, the lipid droplet stage was determined for 10 oocytes randomly chosen among the most advanced mode of developing oocytes and the median value was defined as the stage for the individual. Relationship between the lipid droplet stage at induction of ovulation and hatchability of the ovulated eggs was examined in 23 females, in which the hatching rate markedly fluctuated. Stages of females from which good quality eggs were obtained converged around stage 4 (five largest droplets, 55 to 70 μm) at priming with SPE, stage 6 (90 to 110 μm) at injection with MIS, and stage 7 (110 to 130 μm) at ovulation, while those of females from which low quality eggs were obtained showed distributions over broader ranges. Furthermore, 466 females were induced to mature and the lipid droplet stage at induction of ovulation was examined in 13 females from which high quality eggs (hatching rate >80%) were obtained. Most of the 13 females were at stage 3 (40 to 55 μm) or 4 when primed with SPE, at stage 6 when injected with MIS, and at stage 7 when they ovulated. These results suggest that the optimum stages for SPE priming, MIS injection, and ovulation are stages 3 to 4, 6, and 7, respectively, though the optimum stage for priming will be variable if the temperature and/or the time between priming and MIS injection is varied. We conclude that the lipid droplet stage can be used as a sensitive biomarker to show the optimum timing for inducing ovulation by hormonal treatments.

Vitamin content and quality of eggs produced by broodstock injected with vitamins C and E during artificial maturation in Japanese eel Anguilla japonica

This study was conducted to investigate the effects of injection of vitamins C (VC) and E (VE) into broodstock on the vitamin levels of broodstock and eggs and subsequent egg and larval quality in the Japanese eel. Broodstock were allotted to one of three groups; control group, injected with physiological saline; early group, injected with vitamins during the early part of maturation (1st–3rd weeks after initiation of maturation induction); later group, injected with vitamins during the later part of maturation (4th–6th weeks after initiation of maturation induction). Broodstock were injected with an emulsion of VC and VE at the same time as an injection of salmon pituitary extract during the induction of maturation. The emulsion was prepared by mixing α-tocopherol, sodium ascorbate, an emulsifier, and water just before injection. The levels of both vitamins in eggs and broodstock increased following vitamin injection. However, the accumulation of vitamins in broodstock and eggs was higher for VE than for VC. Hatching rate, survival and normality of larvae increased with vitamin treatments. The results of this study showed that VC and VE injection into broodstock during artificial maturation increases the vitamin content of eggs and can improve egg quality in Japanese eel.

Effects of silvering state on induced maturation and spawning in wild female Japanese eel Anguilla japonica

The effects of silvering state of wild female Japanese eels Anguilla japonica on the success of induced maturation and the following spawning were examined. Thirty-eight females, collected in Mikawa Bay, were divided into four stages based on their silvering state: yellow (Y1), late-yellow (Y2), silver (S1) and late silver eels (S2). Despite injections of salmon pituitary extract (SPE) through the standard technique, Y1 and Y2 eels did not respond to the treatment with undeveloped gonad (gonad-somatic index [GSI]: 0.3–0.9), and all these females died by 5 weeks, probably due to an abnormal physiological condition. Most S1 (81%) and S2 eels (100%) matured completely (GSI: 17.8–51.4), and finally spawned successfully (69% for S1, 89% for S2). S2 eels fully matured with oocytes of over 750 μm in diameter by significantly smaller number of injections of SPE (5–6 times) than the case of S1 eels (6–8 times). The amount of eggs released by S2 eels (0.65±0.11 g/fish per body weight [BW]) was significantly larger than those by S1 eels (0.54±0.09 g/fish per BW). There was no difference in fertilization and hatching rates between eggs released by S1 eels and those of S2 eels. These results indicate that the success of induced maturation and spawning in wild female Japanese eels depends on their silvering state, and matured eggs can be obtained efficiently through the use of S2 eels rather than other stages.

Estimation of gonadal status of Japanese female eel Anguilla japonica by a single injection of salmon pituitary extract

Cultivated eels often either lack a part of the ovary (abnormal) or do not have any gonadal tissue (completely sterile). These abnormalities may cause a failure of induction of sexual maturation of female eels. In order to effectively induce sexual maturation and obtain good quality eggs, female eels having normal ovaries should be selected before induction of maturation. The present study examined effects of injection of salmon pituitary extract (SPE) on serum estradiol-17β levels to estimate gonadal status of sexually immature Japanese eels. At the initial stage (just prior to SPE injection), serum estradiol-17β levels were low and no significant difference was observed among eels possessing the different gonad types (normal, abnormal, and sterile), suggesting that gonadal status cannot be estimated by the measurement of serum estradiol-17β levels. A single injection of SPE significantly increased serum estradiol-17β levels in eels possessing normal and abnormal ovaries, but not in sterile eels. Serum estradiol-17β levels significantly increased from 24 h and were high on day 3. These data indicate that eels possessing ovaries can be distinguished from sterile eels by measurement of serum estradiol-17β between 1 and 3 days after a single injection of SPE.

Induction of triploidy by heat shock in the Japanese eel Anguilla japonica

: Artificial reproduction and subsequent improved culture of the Japanese eel Anguilla japonica have not yet been achieved, but are expected in the near future. To allow the development of aquaculture activities, chromosome manipulation techniques are necessary for the genetic improvement of the eel. In the present paper suitable conditions were determined for heat shock treatment for the induction of triploidy. This was done using fertilized eggs obtained from gametes of cultured eels that were induced to mature by repeated injections of salmon pituitary extract, followed by an injection of 17α,20β-dihydroxy-4-pregnen-3-one to induce ovulation in the female eels and by repeated injections of human chorionic gonadotropin in the male eels. Three variables (i.e. timing after fertilization, temperature, and duration of the shock) were varied to maximize the yield of triploid larvae by examining hatching and triploid rates of treated groups. Ploidy was determined by DNA content flow cytometry and/or chromosome counting. The triploid yield was the highest (70%), when a 37°C heat shock was applied for 3 min, 10 min after fertilization. Under such conditions the rate of triploidy reached 70–100% and the rate of hatching was 5.2–24.6% when the control hatching rate was 28.5% on average.

Reprodução induzida da pirapitinga-do-sul, Brycon opalinus (Cuvier, 1819), mantida em condições de confinamento

Este estudo foi conduzido com o objetivo de obter a reprodução da pirapitinga-do-sul, Brycon opalinus, por intermédio da aplicação de hormônios indutores e determinar a distribuição de freqüência dos diâmetros dos ovócitos em fêmeas aptas à indução. Cento e vinte reprodutores apresentando comprimento entre 25,5 e 32,0 cm foram mantidos em um tanque circular de alvenaria, de 10 m de diâmetro, na Estação de Aqüicultura de Paraibuna/CESP. Para determinação das fêmeas aptas à reprodução, retiraram-se amostras de ovócitos com cateter plástico introduzido pela abertura urogenital. Estes ovócitos foram analisados quanto à cor, tamanho, posição da vesícula germinativa (núcleo) e distribuição de freqüência porcentual dos diâmetros. Os machos foram selecionados pela qualidade do sêmen. Vinte e nove fêmeas aptas à reprodução receberam duas injeções intramusculares de EPS (extrato de pituitária de salmão), sendo a 1ª, na dose de 5 mg/kg e a 2ª, de 10 mg/kg, com intervalo de 12 horas. Os machos que apresentavam ao redor de 90% de motilidade espermática e 85% de espermatozóides vivos receberam dose única de 5 UI de hCG/g (gonadotropina coriônica humana), por ocasião da aplicação da 2ª dose nas fêmeas. Os ovócitos das 14 fêmeas que responderam ao tratamento hormonal eram de cor acinzentada ou vinho, e aquelas que apresentaram alta taxa de fertilização mostraram distribuição de freqüência porcentual dos diâmetros com moda igual a 1900 µm. A taxa média de fertilização foi de 90%, e a de eclosão, 40%.

Artificial induction of maturation and fertilization in the Japanese eel, Anguilla japonica

Repeated injections of salmon pituitary extract (20 mg per fish per week) induced vitellogenesis in feminized, cultivated Japanese eels (Anguilla japonica). Oocytes were attained at the migratory nucleus stage after 11 or 12 injections. Addition of 17,20β-dihydroxy-4-pregnen-3-one (DHP) into the incubation medium induced germinal vesicle breakdown (GVBD) in the oocytes at the migratory nucleus stage. An injection of DHP (2 µg g-1 BW), given 24h after an injection of salmon pituitary extract (20 mg fish-1), succeeded in inducing maturation and ovulation in females which contained occytes at the migratory nucleus stage. Most fish ovulated 15–18h following the DHP injection. Eggs that were ovulated within 15h after the DHP injection showed high fertility and hatchability, but eggs ovulated 18 or 21h after the DHP injection, showed considerably lower fertility and hatchability. A delay between ovulation and stripping of the eggs rapidly decreased both the fertility and hatchability within 6–9h after ovulation, indicating that artificial fertilization must be carried out immediately after ovulation. Repeated injections of human chorionic gonadotropin (hCG) at a concentration of 1 IU g-1 BW week-1 induced spermatogenesis, spermiation, and the acquisition of potential for sperm motility in cultivated males. Most males spermiated after the fifth or sixth injection of hCG, and the milt weight gradually increased and remained constant (1–2 g) from the 11th to 31th injection. Sperm motility peaked 24h after each weekly injection, and decreased from the 3rd day after the injection. Potassium ions are an essential constituent for the maintenance of motility in the eel spermatozoa. Artificial seminal plasma containing 15.2 mM KCl is applicable as a milt diluent. Using these techniques developed for female and male eels, we have succeeded in obtaining many fertilized eggs from cultivated eels.

Changes in fertilization and hatching rates with time after ovulation induced by 17, 20β-dihydroxy-4-pregnen-3-one in the Japanese eel, Anguilla japonica

This study examined the time to ovulation in artificially matured Japanese eel following an injection of 17, 20β-dihydroxy-4-pregnen-3-one (DHP), and changes in fertilization and hatching rates at various times after ovulation. Thirteen females, showing body weight indices (body weight/initial body weight × 100) of 119 ± 2.3% with oocytes at the migratory nucleus stage after 9–12 weekly injections of salmon pituitary extract (20 mg pituitary powder /fish/week), received an injection of salmon pituitary extract (20 mg/fish) followed 24 h later by a DHP injection (2μg/g BW) for induction of ovulation. Eleven of 13 females ovulated within 23 h of the final injection; two, seven, and two females ovulated at 14–17 h, 17–20 h and 20–23 h after the final injection, respectively. The remaining two did not ovulate within 26 h of the injection. Eggs from three out of the 11 ovulated females showed relatively high percent fertility (89.6-39.5%) and hatching rates (47.6-15.3%) when they were fertilized after the first detection of ovulation. Both percent fertilization and hatching rates of eggs retained in the body cavity decreased rapidly within 6–9 h after ovulation. These results indicate that ovulation induced by DHP treatment occurs after a short but well-defined period of time, and that artificial fertilization must be carried out immediately after ovulation in order to obtain good quality eggs.

Philomena oncorhynchi: Effect of hormones on maturation in anadromous sockeye, Oncorhynchus nerka

Adult sockeye salmon, Oncorhynchus nerka (Walbaum), treated with salmon pituitary extract in July survived for up to 47 days and developed nuptial colours in both male and female fish during this period, whereas no such characteristics appeared in untreated control fish. Furthermore, results showed that 20 of 50 adult female Philomena oncorhynchi recovered from the 15 treated fish contained fully developed tailed larvae in the uterus as compared with only one of 44 female P. oncorhynchi from 31 untreated control fish. None of 51 worms from 13 stilboestrol treated adult sockeye showed development in utero beyond an elongated embryo, nor did the host fish develop nuptial colours. P. oncorhynchi and its immature sockeye host (approx. 1 year prior to spawning) remained unaffected by any of three separate hormone treatment experiments using injection of salmon pituitary extract, injection of 17-β-estradiol, and stilboestrol mixed in the food.

Hormone-induced Ovulation of Coho Salmon (Oncorhynchus kisutch) Held in Seawater and Fresh Water

Coho salmon (Oncorhynchus kisutch) mature and spawn naturally in fresh water. We investigated the feasibility of accelerating maturation and/or ovulation of coho salmon retained in seawater during the spawning season, and compared accelerated ovulation of coho salmon in seawater and in fresh water. Effective inducers of ovulation in salmon held in fresh water were partly purified or purified salmon gonadotropin followed 3 d later by luteinizing hormone releasing hormone analogue (LH-RHa); one injection of salmon pituitary extract (SPE); chum salmon pituitary (PIT) followed at 3 d by PIT; and one injection of 17α-hydroxy-20β-dihydroprogesterone. One injection of chinook SPE; coho SPE followed at 3 d by coho SPE; or partly purified or purified salmon gonadotropin followed at 3 d by LH-RHa were effective but to a much lesser extent in those salmon held in seawater. PIT followed at 3 d by PIT, or one injection of 17α-hydroxy-20β-dihydroprogesterane were ineffective in acceleration of maturation/ovulation of coho salmon retained in seawater. Cortisone or 20β-hydroxyprogesterone, which were only tested in salmon in seawater, also failed to elicit early ovulation compared with controls. We conclude that ovarian responses to the treatments of fish retained in seawater are reduced by osmoregulatory difficulties or high adult mortalities. Thus acceleration of ovulation of coho salmon held in seawater is not feasible at this time and requires further research.Key words: coho salmon, spawning, ovulation, induced breeding, gonadotropin, luteinizing hormone releasing hormone analogue

Induced ovulation in coho salmon ( Oncorhynchus kisutch). I. Further studies on the use of salmon pituitary preparations

The work presented extends previous studies on the use of pituitary preparations to induce ovulation in coho salmon. It has been demonstrated that coho salmon may be successfully induced to ovulate up to 2 months prior to their normal peak spawning period and that the use of a single injection of salmon pituitary extract (SPE) to induce ovulation is possible. In November 1977, the experimental fish were divided into six groups. Three groups were administered injections of 0.1 mg/kg partially purified salmon gonadotropin (SG-G100) followed 3 days later by either 2.0 mg/kg SG-G100, 25.0 SPE or 50.0 SPE. A fourth group was administered 10.0 mg/kg SPE followed 3 days later by 50.0 mg/kg. The fifth group received a single injection of 100.0 mg/kg SPE. A control group was injected with saline. All treated groups ovulated significantly earlier than the control. Egg survival and mean egg diameter were within the normal range for coho from Big Qualicum. In September and October 1978, the 10.0 mg/kg SPE primer followed by a 50.0 mg/kg secondary SPE injection were compared to a saline control. In addition, in September a second test group was administered the same primer but was followed by three daily 25.0 mg/kg SPE injections. All treatment groups ovulated significantly earlier than their controls. However, only the 10–50 SPE treatment groups reached cumulative ovulation levels comparable to the controls. Further, these levels increased with closer proximity to the normal spawning period from approximately 40.0% in September to approximately 80% in October. Egg survival and mean degree days to the eyed stage and to hatching for the October experiment fell within the normal range for the Big Qualicum stock. In November 1978, four groups of seven fish received single intraperitoneal or intramuscular injections of either 33.0 mg/kg or 66.0 mg/kg SPE. A control group was injected with saline. All groups ovulated significantly earlier than the control. No significant differences were found between test groups related to dosage level or route of injection. Egg survival and mean degree days to the eyed stage and to hatching fell within the normal range for this stock.