Induction of ovulation in the loach (Paramisgurnus dabryanus) using pimozide and [D-Ala6, Pro9-N-ethylamide]-LHRH

The effects of intraperitoneal injections of [D-Ala⁶, Pro⁹-N-ethylaminde]-luteinizing hormone releasing hormone (LHRH-A) and pimozide (PIM), a dopamine receptor antagonist, on ovulation in loach were investigated. LHRH-A and PIM administered separately were ineffective in inducing ovulation. However, injections of PIM and LHRH-A simultaneously or the injection of PIM 2.5–3 h prior to LHRH-A were highly effective means of inducing ovulation. The simultaneous injection of PIM (1.0 μg/g body wt) and LHRH-A (0.05 μg/g body wt) resulted in a greater ovulatory response than injection of carp pituitary extract (1 pituitary/fish).     

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 17,20-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 3^rd 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.     

Effects of Seasonal Variations, Thyroid and Steroid Hormones, and Carp Pituitary Extract on the Artificial Production of Channel Catfish Ictalurus punctatus× Blue Catfish I. furcatus Hybrids

The primary objective was to compare thyroid hormones and levels of carp pituitary extract for the artificial production of female Ictalurus punctatus × male I. furcatus hybrid catfish. The effects of different carp pituitary extract dosage rates (5, 6, 9, and 10 mg/kg), carp pituitary extract (6 and 10 mg/kg) supplemented with the thyroid hormones thyroxine (T₄) and triiodothyronine (T₃), or pregnenolone (DHP) were determined for inducing ovulation of female channel catfish, fertilization of channel catfish eggs with male blue catfish sperm, and hatching rate of these embryos. Hormone treatments thyroxine and tri-iodothyronine with carp pituitary extract, carp pituitary extract alone, and pregnenolone with carp pituitary extract used to artificially produce hybrid catfish were not different in terms of ovulation rates, eggs/kg, fry/kg body weight of female channel catfish, fertilization rates, or hatching rates (P > 0.05). These findings suggest that lower amounts of carp pituitary extract may be used to induce spawn of female channel catfish for production of channel-blue catfish hybrids and the addition of thyroid and steroid hormones is ineffective at the rates used in this study.     

Stimulation of spermiation and induction of ovulation in pike (Esox lucius)

Mature northern pike were given various hormonal treatments in March or April in order to stimulate spermiation or to induce ovulation. In males the total amount of sperm collected after treatment increased, in comparison with saline-injected males, by 3–11 times with partially purified salmon gonadotropin (PPSG-activity: half of the highly purified s-GTH; injected at doses of between 5 and 100 μg/kg body weight); 3–6 times with crude carp pituitary extract (0.5–3 mg/kg body weight); and 3–7 times with fresh pike pituitaries (14 and 1.2 mg wet weight/kg body weight). The sperm obtained after hormonal treatment was of good quality. Intracardiac injection of superactive LRH analogue had no effect. In females, PPSG induced 90 and 100% ovulation at the doses of 50 and 25 μg/kg body weight. Dried salmon pituitaries (2.5 mg/kg, equivalent to 50 μg of PPSG) gave 25% ovulation; at 10 mg/kg, 25% complete ovulation was again recorded, but in addition 70% of the females showed oocyte maturation and partial ovulation. Similarly, dried carp pituitary (3 mg/kg) induced only oocyte maturation but no ovulation. The oocytes obtained after hormonal treatment were in general fertile. Intraperitoneal injection of LRH in an emulsified form induced neither oocyte maturation nor ovulation. The lack of effect of LRH analogue is discussed and shows that the use of this compound as a substitute for pituitary preparation is not very promising.     

Effects of pimozide and LHRH-Aa on carp (Cyprinus carpio L.) oocyte maturation and ovulationin vivo

Effects of pimozide (Pim) and [(D-Ala⁶, Pro⁹-NEt) LHRH] (LRH-Aa) on common carp oocytes maturation and ovulationin vivo under laboratory and commercial fisheries farm conditions were investigated.Although injections of Pim and LRH-Aa at the doses of 10 mg and 50 µg/kg body weight respectively, did not increase mGtH levels (66.7–155.8 mg/ml) as much as injections of carp pituitary extract (chh) (382.1 ng/ml), induced GtH levels were high enough to induce ovulation. Changes in the ovary caused by Pim and LRH-Aa were similar to those induced by chh, and Pim injected together with LRH-Aa in a single injection gave the same results concerning ovulation induction as when they were applied separately at 6h interval.     

Inducing oocyte maturation, ovulation and spawning in the African catfish, Heterobranchus longifilis Valenciennes (Pisces: Clariidae), using frog pituitary extract

Abstract. Oocyte maturation, ovulation and spawning were successfully achieved in the African catfish, Heterobranchus longifilis Valenciennes, using frog pituitary extract. During artificial spawning, oocyte maturation and ovulation were induced at a dosage of 7mg per kg of individual fish weight. Latency period varied from 7 h at 29°C to 11 h at 25°C. Dead eggs became opaque between 8 and 10h after fertilization while embryogenesis was found to be temperature dependent. Mean hatching and larval deformity rates were 63·08%± 7·56 and 8·08%± 1·93 respectively. During induced natural spawning, the extract was applied at a dosage of 35 mg per kg of female fish weight. Spawning occurred between 10 and 11 h after hypophysation. Mean hatching and larval deformity rates were 41·38%± 8·48 and 10·00%± 2·00 respectively.     

Artificial spawning of European catfish, Silurus glanis L.: stimulation of ovulation using LHRH-a,Ovaprim and carp pituitary extract

Ovulation was stimulated in four groups of European catfish, Silurus glanis L., using injections of des-Gly¹⁰, [D-Ala⁶]-LHRH Ethylamide (20 μg kg^–1) and pimozide (10 mg kg^–1), Ovaprim (0.33 mL kg^–1), and carp pituitary extract (4 mg kg^–1, in one or two doses). A higher percentage of ovulating females (producing eggs of sufficient quality) was found after the LHRH-a and Ovaprim treatments (100% and 80%) in relation to fish treated with the pituitary extract (60% and 66.67%). The greatest weight of eggs was obtained in the case of repeated hypophysation and LHRH-a (1299.69 and 1298.57 g, respectively), and the smallest after single hypophysation (1144.08 g). After 60 h of incubation, the best quality of eggs was found in the group treated with Ovaprim (62.9% of live embryos) and the poorest in the two groups which underwent hypophysation (50.41% and 50.75%). No statistically significant effect by the ovulation stimulators on the characteristic qualitative and quantitative traits of obtained eggs was ascertained.     

Hormone-induced spawning of the Australian freshwater fish Murray cod, Maccullochella peeli (Mitchell) (Percichthyidae)

Murray cod, Maccullochella peeli, originally captured from the wild, underwent normal gonadal development in earthen ponds. Handling of broodfish in the 3 months before a breeding season caused atresia and resorption of oocytes in most females. Cod were removed from the ponds when the water temperature reached 20°C during spring, and final oocyte maturation and ovulation were induced in mature females by injecting 1000 or 2000 IU/kg human chorionic gonadotrophin (HCG) or 2–5 mg/kg of a preparation of the pituitary gland from common carp (CPG). Control treatments and dosages of 100–750 IU/kg HCG did not induce ovulation. Broodfish were held at 21 ± 1°C in 2000-l tanks after injection. The time of stripping and fertilization of Murray cod eggs was an important factor determining their hatchability. There was generally high post-fertilization mortality of eggs stripped within 1 h or between 4 and 6 h of ovulation, but high hatchability of eggs stripped 2–3 h after ovulation. The mean hatchability of eggs stripped 48.5–49.5 h after the injection of 1000 IU/kg HCG was 79.8%, but there were significantly lower mean hatchabilities of eggs stripped after 46–48 h and 50–52 h, as well as after the injection of 2000 IU/kg HCG. Results using CPG were variable. Possible reasons for the high post-fertilization mortality of Murray cod eggs are discussed, and techniques for broodfish handling, injection, stripping and the fertilization and incubation of eggs are presented.     

Artificial spawning of female Polish line 3 carp (Cyprinus carpio L.) after treatment with pituitary homogenate and/or Ovopel

Stimulation of ovulation was carried out in female carp (Cyprinus carpio L.) of Polish line 3, with carp pituitary homogenate, Ovopel (the preparation contains d ‐Ala⁶, Pro⁹NEt‐mGnRH and metoclopramide), and Ovopel and carp pituitary homogenate. The effect of the ovulation stimulators on the weight of eggs expressed in grams and as a percentage of female body weight was determined. The effect of the treatments on the quality of eggs expressed as the percentage of living embryos after 24‐ and 36‐h incubation was also determined. The best results were obtained after stimulation with Ovopel (the priming dose) and pituitary homogenate (the resolving dose). In this case all the females spawned, and the highest yield and quality of eggs were recorded. The poorest results were noted if the ovulation was stimulated with repeated injection of carp pituitary homogenate. The females treated with carp pituitary homogenate gave eggs of poorer quality than those stimulated with two doses of Ovopel or with Ovopel and pituitary homogenate. The percentage of living embryos after 24‐ and 36‐h incubation of eggs was significantly higher for the group stimulated with Ovopel and pituitary homogenate than for the other treatments.     

Artificial spawning of European catfish Silurus glanis L.: differences between propagation results after stimulation of ovulation with carp pituitary and Ovopel

The effect of spawning on European catfish females with an average body weight of 5.6 kg and 11.2 kg was investigated, with carp pituitary and Ovopel being used as ovulation stimulators. Ovopel induced ovulation in a higher percentage of females of both smaller and larger body weight. The applied ovulation stimulators did not significantly affect the weight of the obtained eggs. On the other hand, the effect of the stimulators was highly significant (P≤ 0.01) with respect to the percentage of live embryos after 24‐, 48‐ and 56‐h incubation. The fish of greater body weight yielded eggs of a significantly (P≤ 0.05) greater weight, although of poorer quality. The interaction between the ovulation stimulator and female body weight was statistically significant (P≤ 0.05) for the weight of the obtained eggs and highly significant (P≤ 0.01) for the percentage of live embryos after 48‐h incubation. For the percentage of live embryos after 24‐ and 56‐h incubation, the statistical significance (P≤ 0.05) of this interaction was determined. In the case of females of smaller body weight, a statistically significant (P≤ 0.05) phenotypic correlation was found between the percentage of live embryos after 24‐h incubation and that after 48‐h incubation, and also between the percentage of live embryos after 24‐ and 56‐h incubation.     

Artificial spawning of carp, Cyprinus carpio (L.)

The effect of reproduction was investigated on females of Hungarian strain W, French strain F, and their cross‐breed 1X whose ovulation was stimulated with carp pituitary (0.3 mg kg^?1and, after 12 h, 2.7 mg kg^?1) or Ovopel (one‐fifth of a pellet per kg and, after 12 h, one pellet per kg). It was found that in the case of Ovopel treatment, the percentage of spawning females of strain F and the cross‐breed 1X was higher than in the hypophysed fish compared. The applied ovulation stimulators did not significantly affect the weight of obtained eggs, whereas the significant (P ≤ 0.01) effect was recorded with respect to the quality of eggs after 12‐, 24‐, 36‐ and 48‐h of incubation. After Ovopel stimulation, the quality of eggs was better. The origin of the females had no statistically significant effect on the weight of eggs although the yield of eggs from fish of strain W was much smaller than that from females of strain F and the 1X cross‐breed. The interaction between the ovulation stimulator and the provenance of the females was significant (P ≤ 0.05) for the percentage of live embryos after 48‐h of incubation of eggs. Eggs of the best quality (and highest weight) were obtained from fish of strain F and cross‐breed 1X treated with Ovopel. In females of strain F that spawned within 6 and 10 h after the second Ovopel injection, the effect of the ovulation time on the weight of eggs was non‐significant. It was significant with respect to the percentage of egg fertilization and of live embryos after 36‐h of incubation (P ≤ 0.01 and P ≤ 0.05 respectively). The better quality of eggs (and their higher weight) was recorded when this time was shorter.     

Induced breeding of the South American catfish, Rhamdia sapo (C. & V.)

Pituitary suspensions from the characin, Prochilodus platensis, were injected intraperitoneally into male and female Rhamdia sapo at doses between 0.37 and 6 mg dry weight per kg of body weight. Doses from 0.75 to 6.0 mg/kg effectively induced ovulation. The latent period between injection and ovulation for females held at different temperatures (17 to 27°C) decreased with increasing temperature.Stripping of gametes from both sexes, and dry method fertilization were successful and produced viable eggs. The period between ovulation and stripping for the highest hatching rates decreased from about 9 h at 20°C, to 5 h at 24°C. No viable eggs remained in samples obtained after 15 h at 20°C, or 8 h at 24°C. Hatching percentage and the fraction of deformed fry were negatively correlated (r = ?0.75; P < 0.001).     

Induced Breeding of Two Clariid Catfishes Clurius gariepinus and Heterobranchus bidorsalis Using Tilapia Pituitary Extracts

Induced breeding trials of two clariid catfishes, Clarias gariepinus and Heterobranchus bidorsalis , were conducted using five doses (2, 4, 6, 8 or 10 mg/kg) of acetone-dried tilapia pituitary (ADTP) extracts. Oocyte maturation and ovulation were induced in female catfishes by single intramuscular injection of 6–10 mgkg ADTP; optimum results were obtained with 8 mgkg in both catfishes. At ambient temperature (27 ± 1 C). ovulation occurred within 14–18 h post-injection resulting in 16–20% increase in egg diameter. Fertilization and hatching percentages increased ( P < 0.05) with increases in hormone dosage. Survival of fry fed a mixed zooplankton diet was high (ranging from 79% to 85%) after 30 d of rearing.     

Application of controlled-release,GnRHa-delivery systems in commercial production of white bass X striped bass hybrids (sunshine bass), using captive broodstocks

Hatchery-produced white bass (Morone chrysops) and striped bass (M. saxatilis) reared to maturity in a commercial aquaculture facility, were successfully spawned using controlled-release delivery systems containing the gonadotropin-releasing hormone analog DAla⁶, Pro⁹[NEt]-GnRH (GnRHa). Two-year-old white bass females (mean weight, 0.81 kg) were implanted with different polymer-based, GnRHa delivery systems at doses ranging from 40 to 89 μg GnRHa kg⁻¹ body weight. GnRHa treatment on 20 February 1994, when females contained oocytes up to 720 μm in diameter, induced ovulation of all fish between 35 to 82 h after treatment. The white bass eggs produced were fertilized with sperm from striped bass for the production of sunshine bass. An average of 294500 eggs kg⁻¹ were produced, with a mean fertility of 81.2%, 24 h survival of 46.5%, and overall hatching success of 45%. Survival from hatch to 30 days post-hatch was 78% and the fry weighed between 0.07 and 0.1 g. Overripening of eggs began within 1 h from ovulation and maximum fertilization (60%) was observed when eggs were stripped 0.5 h after ovulation. Fertilization success decreased thereafter to 31% and 10% by 1 h and 3 h after ovulation, respectively. Control fish not treated with GnRHa did not show any signs of final oocyte maturation during the period of the study. GnRHa administration via controlled-release delivery systems appears to be a very effective method for inducing high fecundity ovulation of captive white bass broodstocks, and producing eggs of high fertility and hatching success.     

Induced Ovulation and Spawning in the Catfish,Clarias isheriensis,Using Pituitary Extracts from Non-piscine Sources

Acetone-dried pituitary extracts obtained from non-piscine sources- the common toad, Bufo regularis; the African bullfrog, Rana adspersa; and chicken, Gallus domesticus, were evaluated as alternative inducement agents to piscine-sourced pituitary extracts to spawn Clarias isheriensis. A single dose of 4 mg of dry pituitary extract per kilogram of female was injected intraperitoneally, and it induced ovulation and spawning in all treatments; there was no significant difference (P > 0.05) in percent fertilization of eggs. All pituitary extracts were effective in inducing ovulation and spawning in C. isheriensis and, therefore, can be used as alternative sources of pituitary hormones.     

Ovulation rate,latency period and ova viability after GnRH- or hCG-induced breeding in the Asian catfish Pangasius hypophthalmus(Siluriformes,Pangasiidae)

Over a 3-year period at the Sukamandi station (West Java, Indonesia), 107 Pangasius hypophthalmus females were selected on the basis of a modal oocyte diameter greater than 1.0 mm and treated with either Ovaprim (n = 97) or hCG (n = 10) to induce oocyte maturation and ovulation. The two hormonal treatments led to similar results in terms of ovulation rate (88 and 90 %), hatching rate (72 ± 25 and 82 ± 11 %) and relative fecundity (171 000 ± 73 000 and 128 000 ± 60 000 ova·kg^–1, with Ovaprim and hCG, respectively). The latency period between the last hormone injection and ovulation was negatively correlated to water temperature but showed important variations at a same temperature depending on individual females (e.g. between 5 and 11 h at 28–29 °C). The ovulation time was therefore difficult to predict accurately in this species. The assessment of the viability of ova retained in the ovarian cavity after ovulation showed that the process of overripening occurs rapidly in P. hypophthalmus. The overall quality of ova began to decline as early as 2 h after ovulation and, after 3 h, hatching rates decreased and the proportion of deformed larvae increased significantly in comparison to those observed at the time of ovulation. In some individual females this process occurred even more rapidly, with a sharp decrease in hatching rates between 1 and 2 h post-ovulation. The duration of ova survival did not appear to depend on the type of hormone treatment used to induce ovulation (Ovaprim or hCG). For optimized gamete management in hatcheries, it is therefore recommended to check carefully the females for the occurrence of ovulation (between 3 and 11 h after the last hormone injection, depending on water temperature) and to strip and fertilize the eggs less than 2 h thereafter.     

Ovulation induction in northern pike Esox lucius L. using different GnRH analogues, Ovaprim, Dagin and carp pituitary

The traditional GnRH analogue treatments applying the chemicals in pure form proved to be ineffective in inducing ovulation in northern pike (Esox lucius L). Neither mGnRHa ([D‐Ala⁶, Pro⁹NEt]‐mGnRH) nor sGnRHa ([D‐Arg⁶, Pro⁹‐Net]‐sGnRH) administered alone or together with pimozide (mGnRHa), metoclopramide (mGnRHa) or domperidone (sGnRHa) induced ovulation in females, whereas in groups receiving a carp pituitary injection most females ovulated. Spawning‐inducing agent Dagin did not induce ovulation, whereas all but one female ovulated in the carp pituitary‐treated group. Treatment with another preparation, Ovaprim, resulted in similar or lower ovulation ratio than treatment with carp pituitary. After the Ovaprim treatment, time to ovulation was not as predictable as after the carp pituitary injection. The mean fertilization rate was relatively low and similar in the groups treated with Ovaprim (54.7 ± 12.3% and 58.7 ± 19.1% for the first and second experiment respectively) and with carp pituitary (53.7 ± 10.5% and 58.9 ± 14.9%). The mean pseudogonadosomatic index (PGSI) was also similar between the Ovaprim‐treated group (14.5 ± 6.1%) and the carp pituitary‐treated one (17.9 ± 4.1%). In the present experiments, treatment with Ovaprim was less effective than that with carp pituitary.     

17α-hydroxy-progesterone-induced breeding of the African catfish, Clarias gariepinus (Burchell), without priming with gonadotropin

Female catfish (Clarias gariepinus), raised from eggs to maturity under hatchery conditions and kept at 25°C under a natural photoperiod, were injected with 17α-hydroxy-progesterone in order to induce oocyte maturation and ovulation. This was achieved using dosages of 8 μg/g body weight, administered in two successive injections of 3 and 5 μg/g, with a time interval of 4 h. Eggs could be stripped within 12.5 h after administration of the first injection. Most of the eggs could be fertilized and hatched normally.     

A new tool for induced spawning: The use of 17α-hydroxy-20β-dihydroprogesterone to spawn carp at low temperature

The efficiency of different hormonal treatments to induce ovulation of carp at low temperatures (13–15°C) was tested. “Priming” with a low dose (0.6 mg/kg) of carp pituitary extract was found necessary for a subsequent successful treatment with 17α-hydroxy-20β-dihydroprogesterone (17α-20β P) (2 mg/kg) 1 day later. The eggs produced by this method showed satisfactory fertilization (75–96%) and hatching success (70%).On the other hand, normal hypophysation (5.4 mg/kg) following priming resulted only in partial ovulation and oocyte resorption at this temperature.Priming was shown to increase the gonadotropin level in plasma from about 2 to 35 ng/ml and to induce germinal vesicle migration toward the periphery of oocytes. Neither 17α-20β P nor desoxycorticosterone alone or in association gave any positive result in the absence of a preliminary priming.     

长吻鮠人工催产及脑垂体促性腺激素细胞的超微结构的变化

通过对催产和未催产的长吻鮠脑垂体中腺垂体促性腺激素分泌细胞（ＧＴＨ 细胞） 的分泌活动分析,证实了用促黄体素释放激素类似物（ＬＲＨ－ Ａ） ５０μｇｋｇ 加ＤＯＭ５ｍｇｋｇ 混合注射催产长吻鮠,能有效地促使ＧＴＨ细胞分泌促性腺激素,诱导卵母细胞成熟和排卵,催产效果显著。超微结构的进一步观察,揭示了长吻鮠脑垂体ＧＴＨ 细胞中存在两种分泌颗粒,即分泌小球,直径１２００～２０００ｎｍ ,电子密度低;分泌颗粒直径３００ ～５００ｎｍ ,电子密度高。分泌小球释放与卵母细胞的发育成熟有关,分泌颗粒的释放则与排卵相关。