L.W. Crim

Advancement and synchrony of ovulation in Atlantic salmon with pelleted LHRH analog

Abstract The capacity of long-acting, pelleted LHRH analog to advance and synchronize spawning in the female Atlantic salmon was examined at two different times prior to spawning. Treatment with LHRH analog 45 days prior to the time of normal spawning induced early spawning in 30% of the females despite a significant elevation of circulating gonadotropin in all treated fish. When LHRH analog treatment was administered approximately 28 days prior to the control spawning time, ovulatory levels of gonadotropin rapidly appeared in the serum, and 94% of the females were spawned within 9 days of the hormone implantation. A detrimental effect upon egg quality was observed following early induction of spawning in September. Egg survival was low in the case of some treated females but in others receiving LHRH analog, egg survival was comparable to controls. It is concluded that pelleted LHRH analog may be used to advance and synchronize ovulation in Atlantic salmon, especially in ripened females nearing the time for natural spawning.

Motility, fertility and ultrastructural changes of ocean pout (Macrozoarces americanus L.) sperm after cryopreservation

Abstract The present study examined the possibility of long term storage, by cryopreservation in liquid N 2 , of the sperm of ocean pout ( Macrozoarces americanus L.), an internally fertilizing marine fish, and the changes in motility, fertility and ultrastructure of the sperm after freeze and thaw. Four cryoprotectants, including dimethyl sulfoxide (DMSO), and three semen diluents (A, B and C) were tested for their influence on sperm motility. Since the fresh sperm displayed the highest motility in diluent C, which had the closest chemical composition to that of the seminal plasma of ocean pout, with DMSO, this solution (C-DMSO) was chosen as a diluent for the present study of ocean pout semen cryopreservation. Fresh semen was diluted in three volumes of C-DMSO and filled in 0.25- or 1.7-ml straws, then frozen in liquid N 2 vapor. When the internal temperature of the straws had dropped to −95°C, the straws were plunged into the liquid N 2 . To recover the sperm motility, the frozen semen was thawed in 1 or 30°C water bath for 30 and 7 s, respectively. It was demonstrated that the presence of DMSO in semen extender was essential for protecting the sperm from dying during freeze and thaw, and 20% of DMSO (v/v) yielded the highest post-thawed sperm motility (20–25% of the total cells). A freezing rate of average 9°C/min during the initial freezing phase (in liquid N 2 vapor) produced a higher post-thawed sperm motility than that produced by faster (18°C/min) and slower (6°C/min) freezing rates. Thawing the frozen semen in 30 or 1°C water did not cause any difference in terms of sperm motility. The loss of sperm motility during freeze and thaw might be due to the ultrastructural changes of sperm, e.g., severe swelling of the mitochondria or dehydration of cytoplasm at the midpiece, revealed by scanning electron microscope. The motile sperm in the post-thawed semen possessed fertility because in vitro artificial insemination of fresh eggs using the post-thawed semen yielded a fertilization rate of 33% vs. 48–58% from fresh semen.

Administration of LHRH analogues in various ways: effect on the advancement of spermiation in prespawning landlocked salmon, Salmo salar

Abstract During an experimental period of 12 days, the onset of spermiation, quantity of collectable milt, spermatocrit, plasma and pituitary gonadotropin hormone (GtH) were compared in control and LHRH analogue (LHRHa)-treated prespawning landlocked salmon. Des-Gly 10 -[D-Ala 6 ]-LHRH ethylamide (D-Ala 6 -LHRH EA) was administered either by intraperitoneal multiple injections in saline or 40% propylene glycol or by a single intraperitoneal implant in silicone rubber. [6-(D-2-naphthylalanine)] LHRH (D-Nal (2) 6 -LHRH) was administered in a single cholesterol pellet. All the methods of administering LHRHa effectively accelerated the onset of spermiation and increased the volume of collectable milt. Study of the spermatocrit indicated that this increase is associated with an advancement in the production of spermatozoa and is not just due to sperm dilution. Furthermore, this increase in milt volume is related to an increase in plasma GtH. On the other hand, LHRHa treatments did not provoke any change in pituitary GtH content.

Accelerated ovulation by pelleted LHRH analogue treatment of spring-spawning rainbow trout (Salmo gairdneri) held at low temperature

Abstract Pelleted LHRH analogue formulated for long term hormone delivery was administered by two routes to maturing, spring spawning, female rainbow trout in an attempt to accelerate spawning. At water temperatures ranging from 0.5–2.0°C, spawning was advanced by approximately 27 days. Rapid increases in the level of plasma gonadotropic hormone at 2 weeks indicates that pituitary stimulation occurred well in advance of ovulation, despite low temperatures. Egg diameter and fertility were comparable in sham control and experimental fish. Heavy mortalities occurred in eggs taken from accelerated spawners, however, eggs from 35% of these females were still viable at the eyed stage. Although further studies directed at egg quality appear warranted, pelleted LHRH implants could prove useful in advancing spawning time in order to meet schedules of smolt production or assist in synchronizing the spawning time of salmonid brood stock reared or held in salt water.

Induced spawning of sea bass, Lates calcarifer, and rabbitfish, Siganus guttatus, after implantation of pelleted LHRH analogue

Abstract Captive Lates calcarifer broodstock at Tigbauan, Iloilo (Philippines) were implanted with cholesterol-based pellets of the LHRH analogue D-Trp6-desGly10-LHRH ethylamide or D-hArg(Et2)6,Pro9-NHet-LHRH at doses between 9.0 and 23.5 μg/kg body weight. In May, one of ten LHRH-treated females released partially hydrated ova into the tank 4 days after implantation. In July, at least one (and probably four) of five LHRH-treated females spawned in the tank 2 days after implantation; 2.6 million hatchlings were collected. In August, both LHRH-treated females spawned in the tank 2 days after implantation; 978 000 hatchlings were collected. None of the sham-operated control fish spawned in any of the experiments. Captive Siganus guttatus broodstock implanted with silastic-based pellets of the LHRH analogue D-Nal (2)6 LHRH spawned 1–2 days earlier than sham-operated controls.

The influence of LHRH analog on oocyte development and spawning in female Atlantic salmon, Salmo salar☆

Abstract A study of [D-Nal(2) 6 ]LHRH analog implantation in female Atlantic salmon was conducted to determine the capacity of sustained releasing hormone treatment to stimulate ovary growth and induce spawning. Judging the reproductive status of individual females at the beginning of hormone treatment was accomplished by ovary biopsy techniques allowing in vitro oocyte analysis. The gonadotropin and steroid hormone patterns associated with oocyte development and spawning in the Atlantic salmon were determined from blood sampling throughout the study period. We conclude that implanted LHRH analog stimulates development of the oocyte during the vitellogenic phase of reproduction; it is again demonstrated that LHRH analog treatment is an effective tool for synchronizing spawning in the Atlantic salmon.

Induced spawning of maturing milkfish (Chanos chanos Forsskal) with gonadotropin-releasing hormone (GnRH) analogues administered in various ways☆

Abstract The response of mature female captive milkfish to mammalian and salmon gonadotropin-releasing hormone analogues (mGnRH-A and sGnRH-A) was investigated. Prior to spawning, six groups of three females received (1) 10–16 μg mGnRH-A from an osmotic pump implanted intraperitoneally (IP); (2) 100 μg mGnRH-A from a cholesterol/cellulose pellet implanted IP; (3) 10 μg/kg mGnRH-A as an intramuscular (IM) injection; (4) 10–16 μg sGnRH-A from an osmotic pump implanted IP; (5) 100 μg sGnRH-A from a cholesterol/cellulose pellet implanted IP, and (6) a cholesterol/cellulose pellet without analogue implanted IP. The most effective treatment was 100 μg sGnRH-A/fish given in a cholesterol/cellulose pellet; all ( 3 3 ) of the fish spawned. However, mGnRH-A was more effective ( 2 3 ) compared with sGnRH-A ( 1 3 ) if osmotic pumps were used to administer GnRH-A. If the dose and method of administration were not considered, then the salmon and mammalian GnRH analogues were equally effective (62–67%) for induction of ovulation and natural spawning in milkfish. Gonads of control fish regressed. At the doses tested, injections or pellet implantations were more effective compared with osmotic pumps. All pellet-implanted and injected females responded to treatment and 75% ( 6 8 ) spawned; half ( 3 6 ) of the pump-implanted females spawned. Spawning occurred from 18 to 36 h after treatment.

Direct feminization of lumpfish (Cyclopterus lumpus L.) using 17β-oestradiol-enriched Artemia as food

Abstract Artemia nauplii cultured in lipid-enriched media containing 0, 5, 10 or 20 mg 17 β -oestradiol/l for 24 h contained 0, 140, 90 and 231 ng 17 β -oestradiol/mg dry wt., respectively. Feminization of lumpfish larvae was essentially 100% when the larvae were fed Artemia containing 231–407 ng 17 β -oestradiol/mg dry wt. at first feeding for 20 days. Immersing eggs or newly hatched larvae at 22, 29 and 36 days post-fertilization in 200 μ g 17 β -oestradiol/l for 2 h resulted in 68% and 46% females in duplicate treatments. The ovaries of some fish which consumed Artemia containing 17 β -oestradiol were smaller, filiform and less developed than those from similar size control females although no spermatogenic tissue was detected in these filiform ovaries. This may indicate that females with filiform ovaries were feminized, genotypic males. It appears that direct sex-reversal techniques, based upon steroid-supplemented Artemia nauplii, has promise of producing monosex populations in fish larvae requiring a diet of live prey during the period of sex determination.

Induced spawning of maturing milkfish (Chanos chanos) using human chorionic gonadotropin and mammalian and salmon gonadotropin releasing hormone analogues

Abstract The response of maturing female milkfish to D-Ala6-des Gly10 mammalian GnRH ethylamide (mGnRH-A), D-Arg6-des Gly10 salmon GnRH ethylamide (sGnRH-A) and human chorionic gonadotropin (hCG) was investigated. The GnRH analogues and hCG were equally effective when administered by intramuscular injection at doses of 10 μg/kg and 100 μg GnRH-A/fish or 1000 IU hCG/fish. All of the females injected with HCG and 87.5% ( 7 8 ) of females injected with GnRH-A spawned. Pellet implantation of the GnRH analogues, however, was less effective based on 100 μg of pellet per fish, which provided from 20 to 36 μg of analogue per kg fish. Fish implanted with mGnRH-A or sGnRH-A showed responses which varied from oocyte hydration to spawning. Only 3 7 implanted with mGnRH-A and 1 7 implanted with sGnRH-A spawned; in the latter group, the average egg diameter was 11–17% smaller at the time of treatment compared with the other treated groups. Except for one, all fish with egg diameters above 0.65 mm had hydrated/ovulated oocytes or spawned. Females which spawned had egg diameters above 0.71 mm.

Effects of LHRH and LHRH-A on plasma GtH levels and maturation/ovulation in the common carp, Cyprinus carpio, kept under various environmental conditions

Abstract Experiments were carried out to determine if some environmental factors usually present in spawning ponds at the time of ovulation could potentiate the effects of LHRH or LHRH-A on the plasma level of GtH, oocyte maturation or ovulation in the common carp, Cyprinus carpio . In one experiment females were held in the absence of a male in cages placed in rearing ponds containing a dense population of unicellular algae; the cages touched the bottom but there was no vegetation inside the cages. When LHRH-A was given in two injections (the first 5 μg/kg, the second, 6 h later, 50 μg/kg) plasma GtH was increased and followed oocyte maturation. Ovulation occurred in 50% (3 of 6) of the females. In another experiment, using a similar LHRH-A treatment schedule, the females were held in aquaria with added vegetation to serve as a spawning substrate; in such a simplified environment the effect of LHRH-A was not potentiated. Similarly, the natural environment of spawning ponds including the presence of males was of no benefit to the response to low doses of LHRH or LHRH-A when the water temperature was low (12°C). These results suggest that some undetermined environmental factors besides vegetation present in a pond situation may potentiate the effect of LHRH-A, possibly via abatement of the action of an endogenous gonadotropin release-inhibitory factor.