RESUMEN
In northern Japan, juvenile chum salmon Oncorhynchus keta (Walbaum) are released from hatcheries to enhance the fishery resource. Infections with ectoparasitic protozoans, particularly the flagellate Ichthyobodo salmonis and the ciliate Trichodina truttae, occasionally cause severe mortality among hatchery-reared juveniles. This study examined the susceptibility of the two parasites to wide-ranging UV irradiation (experiment 1) and then investigated whether UV disinfection of the rearing water using a commercial device was useful for preventing infections among juveniles in a small-scale rearing system over a 28-day period (experiment 2). In experiment 1, parasite mortality reached 100% with UV irradiation doses of ≥9.60 × 105 µW s/cm2 for I. salmonis and ≥8.40 × 105 µW s/cm2 for T. truttae. In experiment 2, disinfection of the rearing water at a UV irradiation dose of 2.2 × 106 µW s/cm2 succeeded in complete prevention of both parasites in the juvenile salmon. These results elucidate the minimum dose of UV irradiation for inactivation of I. salmonis and T. truttae, and demonstrate the usefulness of water disinfection using a commercial UV irradiation device to prevent infections by these parasites in hatchery-reared juvenile chum salmon.
Asunto(s)
Infecciones por Cilióforos/veterinaria , Desinfección/métodos , Infecciones por Euglenozoos/veterinaria , Enfermedades de los Peces/parasitología , Kinetoplastida/efectos de la radiación , Oligohimenóforos/efectos de la radiación , Rayos Ultravioleta , Animales , Infecciones por Cilióforos/prevención & control , Infecciones por Euglenozoos/prevención & control , Enfermedades de los Peces/prevención & control , Explotaciones Pesqueras , Japón , Oncorhynchus keta/parasitología , Purificación del Agua/métodosRESUMEN
The present study performed three experiments to establish a practical prevention strategy for the ectoparasitic flagellate Ichthyobodo salmonis and ciliate Trichodina truttae in hatchery-reared juvenile chum salmon Oncorhynchus keta using dietary supplementation with oregano essential oil. Experiment 1 showed that a diet supplemented for 3 weeks with 0.02% oregano essential oil significantly prevented infection with I. salmonis and T. truttae in juveniles reared in small tanks. Experiment 2, in outdoor hatchery ponds, demonstrated that the oregano treatment completely prevented I. salmonis infection for 52 days and T. truttae infection for 38 days. Oregano-treated juvenile mortality attributable to infection with these protozoans also decreased to 7.6% of control juvenile mortality, confirming the utility of this treatment in cultured O. keta. Physiological analyses of the oregano-treated juveniles elucidated the treatment's safety in relation to their metabolism, osmoregulation, natural immunity and olfactory responses and also detected carvacrol (a major component of oregano essential oil which shows antimicrobial activity) on the skin. In experiment 3, exposure of the two protozoans to oregano essential oil revealed a weak antiparasitic action on the body surface of the juvenile O. keta. The overall results demonstrate that dietary oregano supplementation is a practical prevention strategy for I. salmonis and T. truttae in hatchery-reared juvenile O. keta and suggest the possibility that its anti-parasitic action is attributable to a component of the oil that emerges onto the skin of the body of the fish.
Asunto(s)
Suplementos Dietéticos , Aceites Volátiles/uso terapéutico , Oncorhynchus keta/parasitología , Origanum , Enfermedades Parasitarias en Animales/prevención & control , Animales , Acuicultura , Dieta/veterinaria , Kinetoplastida , Oligohimenóforos , Fitoterapia , Piel/químicaRESUMEN
Infestations of the ectoparasitic flagellate Ichthyobodo salmonis and the ciliate Trichodina truttae have caused acute mortalities of hatchery-reared juvenile chum salmon Oncorhynchus keta in Hokkaido, northern Japan. This study examined the epizootiology of I. salmonis and T. truttae on wild chum salmon as a possible infection source of the 2 parasitic protozoans in hatcheries. Infestations by both ectoparasites were detected on freshwater-adapted adult and juvenile chum salmon in all 4 rivers examined. This is the first study of an anadromous Pacific salmonid to report infestation of I. salmonis and T. truttae in adults returning for spawning. Among the marine-inhabiting phase of chum salmon, infestation with I. salmonis, but not T. truttae, was observed on adults and juveniles. The 2 protozoans were experimentally transmitted at the same time from wild to hatchery-reared chum salmon juveniles, and caused a high rate of mortality in the hatchery fish. In freshwater, the proliferation rate of T. truttae was greater than that of I. salmonis. These observations show that the euryhaline ectoparasite I. salmonis can infest chum salmon throughout their life cycle, in both river and ocean habitats, whereas T. truttae is able to infest these salmonids only in freshwater. Furthermore, wild chum salmon were shown to be a potential infestation source for both T. truttae and I. salmonis in hatchery fish.
Asunto(s)
Infestaciones Ectoparasitarias/veterinaria , Enfermedades de los Peces/parasitología , Oncorhynchus keta/parasitología , Infecciones Protozoarias en Animales/parasitología , Migración Animal , Animales , Acuicultura , Infestaciones Ectoparasitarias/epidemiología , Infestaciones Ectoparasitarias/parasitología , Enfermedades de los Peces/epidemiología , Japón/epidemiología , Infecciones Protozoarias en Animales/epidemiologíaRESUMEN
Insulin-like growth factor-I (IGF-I) has been shown to be involved in pubertal activation of gonadotropin (GTH) secretion. The aim of this study was to determine if IGF-I directly stimulates synthesis and release of GTH at an early stage of gametogenesis. The effects of IGF-I on expression of genes encoding glycoprotein alpha (GPalpha), follicle-stimulating hormone (FSH) beta, and luteinizing hormone (LH) beta subunits and release of FSH and LH were examined using primary pituitary cells of masu salmon at three reproductive stages: early gametogenesis, maturing stage, and spawning. IGF-I alone or IGF-I + salmon GnRH (sGnRH) were added to the primary pituitary cell cultures. Amounts of GPalpha, FSHbeta, and LHbeta mRNAs were determined by real-time PCR. Plasma and medium levels of FSH and LH were determined by RIA. In males, IGF-I increased the amounts of all three subunit mRNAs early in gametogenesis in a dose-dependent manner, but not in the later stages. In females, IGF-I stimulated release of FSH and LH early in gametogenesis, whereas no stimulatory effects on the subunit mRNA levels were observed at any stage. IGF-I + sGnRH stimulated release of FSH and LH at all stages in both sexes, but had different effects on the subunit mRNA levels depending on subunit and stage. The present results suggest that IGF-I itself directly stimulates synthesis and release of GTH early in gametogenesis in masu salmon, possibly acting as a metabolic signal that triggers the onset of puberty.
Asunto(s)
Gametogénesis/fisiología , Gonadotropinas/metabolismo , Factor I del Crecimiento Similar a la Insulina/farmacología , Hipófisis/efectos de los fármacos , Salmón/metabolismo , Animales , Femenino , Hormona Folículo Estimulante/genética , Hormona Folículo Estimulante/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Gonadotropinas/genética , Hormona Luteinizante/genética , Hormona Luteinizante/metabolismo , Masculino , Hipófisis/citología , Hipófisis/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reproducción/fisiología , Salmón/genética , Estaciones del Año , Maduración Sexual/fisiologíaRESUMEN
Expression of genes encoding gonadotropin (GTH) subunits in the salmon pituitary was regulated by salmon gonadotropin-releasing hormone (sGnRH) and sex steroid hormones in a reproductive stage-dependent manner, probably through DNA-binding transcription factors. Direct effects of these hormones on expression of genes encoding salmon fushi tarazu factor 1 homolog (sFF1-I) and estrogen receptor alpha (ERalpha) were therefore examined by use of primary pituitary cell cultures of masu salmon at different reproductive stages. Pituitaries were collected in March (before initiation of gonadal maturation), in May (early maturing), in July (late maturing), and in September (spawning period). Amounts of sFF1-I and ERalpha mRNAs in the pituitary cells were determined by real-time polymerase chain reactions after a treatment with sGnRH, estradiol-17beta (E2), testosterone (T) or 11-ketotestosterone (11KT). The amounts of sFF1-I mRNA were elevated by E2 in the males, and by sGnRH and T in the females before initiation of gonadal maturation and at the early maturing stage. The amounts of ERalpha mRNA in the early maturing females were elevated by sGnRH. Effects of sGnRH were not significant at the late maturing and spawning stages. The amounts of ERalpha mRNA in the spawning males were halved by 11KT and E2, and those of sFF1-I and ERalpha mRNAs in the late maturing females were decreased by T and 11KT. These results indicated that responsiveness of sFF1-I and ERalpha genes to sGnRH and sex steroid hormones is seasonally variable in relation to reproductive stages. Expression of sFF1 and ERalpha genes should be stimulated at the early stages of gonadal maturation prior to increases in the amounts of GTH subunit mRNAs, while attenuated after the late maturing period when stored amounts of GTH subunit mRNAs reached near the maximum.
Asunto(s)
Receptor alfa de Estrógeno/genética , Factores de Transcripción Fushi Tarazu/genética , Regulación de la Expresión Génica , Hormona Liberadora de Gonadotropina/metabolismo , Gonadotropinas/metabolismo , Salmón/fisiología , Maduración Sexual/fisiología , Animales , Células Cultivadas , Femenino , Masculino , Hipófisis/citología , Subunidades de Proteína , ARN Mensajero/metabolismo , Salmón/genéticaRESUMEN
Effects of insulin-like growth factor I (IGF-I) and salmon gonadotropin-releasing hormone (sGnRH) on expression of gonadotropin (GTH) subunit genes were examined using primary pituitary cell cultures of masu salmon (Oncorhynchus masou). Fishes were assessed at three reproductive stages, i.e., in April (early maturation), in June (maturing), and in September (spawning). Amounts of GTH subunit mRNAs in pituitary cells were determined using real-time PCR after incubation with IGF-I and/or sGnRH. IGF-I alone had almost no effects on three GTH subunit mRNAs in both sexes, except for decrease in follicle-stimulating hormone (FSH) beta mRNA in males in June. sGnRH alone was effective in stimulation of FSHbeta and luteinizing hormone (LH) beta gene expression in males in April. Thereafter it had no significant effects on GTH subunit mRNAs, although in September it tended to increase FSHbeta and LHbeta mRNAs in females. Co-administered IGF-I counteracted the sGnRH-induced expression of FSHbeta and LHbeta genes in males in April, but not in females in September. These results suggest that IGF-I is involved in direct regulation of GTH subunit genes during sexual maturation. In particular, IGF-I differently modulates sGnRH-induced GTH subunit gene expression, depending on reproductive stages.
Asunto(s)
Regulación de la Expresión Génica/efectos de los fármacos , Hormona Liberadora de Gonadotropina/farmacología , Gonadotropinas Hipofisarias/genética , Factor I del Crecimiento Similar a la Insulina/farmacología , Oncorhynchus/fisiología , Hipófisis/citología , Hipófisis/efectos de los fármacos , Maduración Sexual/fisiología , Animales , Células Cultivadas , Relación Dosis-Respuesta a Droga , Femenino , Hormona Folículo Estimulante de Subunidad beta/metabolismo , Masculino , Oncorhynchus/genética , Hipófisis/metabolismo , Subunidades de Proteína/genética , Reproducción/efectos de los fármacos , Reproducción/fisiología , Estaciones del Año , Factores de Tiempo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Expression of genes encoding growth hormone (GH), prolactin (PRL), and somatolactin (SL) in growing and maturing salmon was stimulated by gonadotropin-releasing hormone (GnRH) analog during particular periods of the life cycle. GnRH therefore appears to directly and/or indirectly regulate gene expression for GH, PRL, and SL in combination with the pituitary-gonadal axis, such as sex steroid hormones. Direct effects of salmon GnRH (sGnRH), estradiol-17beta (E2), testosterone, and 11-ketotestosterone (11KT) on the amounts of GH, PRL, and SL mRNAs were thus examined using primary pituitary cell cultures of masu salmon at the four reproductive stages. We also determined the amounts of mRNA encoding pituitary specific POU homeodomain transcription factor (Pit-1) by real-time polymerase chain reactions. The amounts of GH, PRL, and SL mRNAs in the control cells elevated with gonadal maturation, coincidently with those of Pit-1 mRNA. sGnRH at 1.0 nM elevated the amounts of all mRNAs examined in the pre-spawning females, whereas significant effects were not observed with 100 nM sGnRH at any reproductive stages. Sex steroid hormones had no significant effects before initiation of gonadal maturation and at the maturing stage. In the males, E2 tended to decrease the amounts of SL mRNA in the pre-spawning stage. In the females, E2 and 11KT increased the amounts of PRL and SL mRNAs in the pre-spawning stage, but halved those of PRL mRNA in the spawning stage. The amounts of Pit-1 mRNA changed coincidently with those of PRL and SL mRNAs at all examined stages. The effects of E2 alone were abolished by 100 nM sGnRH. The present results indicated that both sGnRH and steroid hormones directly modulate synthesis of Pit-1, and further expression of PRL and SL genes. sGnRH may indirectly regulate GH/PRL/SL family hormone genes through the pituitary-gonadal axis, particularly in the late stage of gametogenesis.
Asunto(s)
Proteínas de Peces/metabolismo , Glicoproteínas/metabolismo , Hormonas Esteroides Gonadales/fisiología , Hormona Liberadora de Gonadotropina/análogos & derivados , Hipófisis/metabolismo , Hormonas Hipofisarias/metabolismo , Salmón/crecimiento & desarrollo , Factor de Transcripción Pit-1/genética , Animales , Células Cultivadas , ADN Complementario/análisis , Estradiol/fisiología , Femenino , Proteínas de Peces/genética , Regulación de la Expresión Génica , Glicoproteínas/genética , Hormona Liberadora de Gonadotropina/fisiología , Hormona del Crecimiento/genética , Hormona del Crecimiento/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Masculino , Datos de Secuencia Molecular , Hipófisis/citología , Hormonas Hipofisarias/genética , Prolactina/genética , Prolactina/metabolismo , ARN Mensajero/metabolismo , Salmón/genética , Maduración Sexual/genética , Maduración Sexual/fisiología , Testosterona/análogos & derivados , Testosterona/fisiología , Factor de Transcripción Pit-1/metabolismoRESUMEN
Effects of salmon gonadotropin-releasing hormone (sGnRH) and estradiol-17beta (E2) on gene expression and release of gonadotropins (GTHs) were examined in masu salmon (Oncorhynchus masou) using primary pituitary cell cultures at three reproductive stages, initiation of sexual maturation in May, pre-spawning in July, and spawning in September. Amounts of GTH subunit mRNAs were determined by real-time polymerase chain reaction, and levels of GTH released in the medium were determined by RIA. In control cells, the amounts of three GTH subunit mRNAs (alpha2, FSHbeta, and LHbeta) peaked in July prior to spawning. FSH release spontaneously increased with gonadal maturation and peaked in September, whereas LH release remained low until July and extensively increased in September. Addition of E2 to the culture extensively increased the amounts of LHbeta mRNA in May and July in both sexes. It also increased the alpha2 mRNA in July in the females. In contrast, sGnRH alone did not have any significant effects on the amounts of three GTH subunit mRNAs at all stages, except for the elevation of alpha2 and FSHbeta mRNAs in July in the females. Nevertheless, synergistic effects by sGnRH and E2 were evident for all three GTH subunit mRNAs. In May, sGnRH in combination with E2 synergistically increased the amounts of LHbeta mRNA in the males and alpha2 mRNA in the females. However, in July the combination suppressed the amounts of alpha2 and FSHbeta mRNAs in the females. sGnRH alone stimulated LH release at all stages in both sexes, and the release was synergistically enhanced by E2. Synergistic stimulation of FSH release was also observed in May and July in both sexes. These results indicate that a functional interaction of sGnRH with E2 is differently involved in synthesis and release of GTH. The synergistic interaction modulates GTH synthesis differentially, depending on subunit, stage, and gender, whereas it potentiates the activity of GnRH to release GTH in any situation.
Asunto(s)
Estradiol/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Hormona Liberadora de Gonadotropina/farmacología , Gonadotropinas/biosíntesis , Oncorhynchus/fisiología , Hipófisis/metabolismo , Desarrollo Sexual/fisiología , Animales , Peso Corporal/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Femenino , Inmunoensayo , Masculino , Hipófisis/efectos de los fármacos , Hipófisis/crecimiento & desarrollo , ARN Mensajero/biosíntesis , Radioinmunoensayo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Conducta Sexual Animal/fisiologíaRESUMEN
To examine the pluripotency of cryopreserved blastomeres, we transplanted them into blastula. Donor blastomeres were prepared from blastula of goldfish (Carassius auratus) and cryopreserved in liquid nitrogen for two months. Fifty-five percent and 44% of blastomeres survived after thawing. Cryopreserved blastomeres were transplanted to the blastula of triploid crucian carp (C. a. longsdorfii), which reproduces gynogenetically in nature. At four days after the operation, resultant chimeric embryos transplanted with cryopreserved blastomeres showed a survival rate (41.6%) lower than that of embryos transplanted with unfrozen blastomeres (57.1%). Transplanted blastomeres were histologically identified in various organs derived from all three germ layers. A primordial germ cell differentiated from a cryopreserved blastomere was detected in one of the 32 chimeric fish examined. These results suggest blastomeres that survive after cryopreservation retain their pluripotency and are able to differentiate into both somatic and germ cell lines.
Asunto(s)
Blastómeros/trasplante , Criopreservación , Carpa Dorada/embriología , Quimera por Trasplante/fisiología , Trasplante Heterólogo/métodos , Animales , Blastómeros/fisiología , Blástula/fisiología , Carpas/fisiología , Técnicas Histológicas , Quimera por Trasplante/anatomía & histologíaRESUMEN
In order to preserve genetic resources of chum salmon, Oncorhynchus keta, optimum conditions for cryopreservation of isolated blastomeres were investigated. Survival rates under various conditions were compared: the nature and the concentration of cryoprotectants before and after freezing, the seeding temperature, and the developmental stages of donor embryos. Isolated blastomeres immersed for 30 min in Eagle's MEM containing both a cryoprotectant and 10% fetal bovine serum (FBS) at 10 degrees C were transferred into a straw and frozen at 1 degrees C/min to -30 degrees C by a programmable freezer before being plunged into liquid nitrogen. Ice seeding was carried out at -5 to -15 degrees C. Frozen blastomeres were thawed in water at 15 degrees C. Blastomeres cryopreserved with MEM containing 10% dimethyl sulfoxide (Me(2)SO) and 10% FBS (10% Me(2)SO/MEM10) showed higher survival rates than those cryopreserved with MEM containing 10% FBS and 10% glycerol, ethyleneglycol, 1, 2-propanediol, or sucrose. Blastomeres treated with 10% Me(2)SO/MEM10 showed higher survival rates than those treated with MEM containing only 10% Me(2)SO. Blastomeres seeded above -10 degrees C showed higher survival rates than non-seeded ones. Frozen blastomeres at advanced stages demonstrated high survival rates. Blastomeres cryopreserved under optimum conditions showed survival rates of 59.3+/-2.8%. These results indicate that 10% Me(2)SO/MEM10 is a suitable cryoprotectant medium to cryopreserve chum salmon blastomeres, that seeding should be carried out above -10 degrees C on pre-freezing, and that blastomeres at the blastula stage should be used as material.