Factors Affecting Cryopreservation Efficiency and Enzyme Activity in Northern Pike,Esox lucius,Sperm

Abstract

Two freezing techniques (straws and pellets), three cryo-protectants (DMSO, glycerol, and DMA) in four concentrations, and several extenders were tested to determine their suitability for cryopreservation of northern pike, Esox iucius, sperm. Activity of aspar-tate aminotransferase (AspAT) and acid phosphatase (AcP) in cryo-preserved milt was determined. Fertilization ability of cryopreserved milt was affected by the freezing technique, by type and concentration of cryoprotectant, as well as by the kind of extender used. These factors also influenced AspAT and AcP activity assayed in cryopreserved sperm. Extender containing 0.6 M sucrose + 15% DMSO + 10% egg yolk was most suitable for cryopreservation of pike sperm in pellet form (90.5% of eyed eggs, as compared to control group, which was 89.1%).     

Cryopreservation of rainbow trout spermatozoa: The influence of sperm quality,egg quality and extender composition on post-thaw fertility

The presence of 5% to 20% hen's egg yolk in a sucrose-based extender significantly improved post-thaw fertility of cryopreserved rainbow trout (Salmo gairdneri) spermatozoa compared to when the extender without hen's egg yolk was used. However, the degree of increased cryoprotection associated with hen's egg yolk was affected by the quality of the milt. Considerable variation was detected in the performance of various batches of trout eggs used to test post-thaw fertility and the composition of the extender was shown to affect fertilizations differentially with some of the eggs. Despite this variation, the extender containing 10% hen's egg yolk consistently gave high post-thaw fertility in samples of cryopreserved milt (67.3±3.0% S.E.M.) in thirty replicated trials. As such, the method described is reliable for cryopreserving rainbow trout milt and fertilizing small quantities of eggs.     

Effect of extender composition and freezing rate on post-thaw motility and fertility of Arctic char, Salvelinus alpinus (L.), spermatozoa

The effects of extender composition and freezing rate on motility and fertility of frozen‐thawed Arctic char, Salvelinus alpinus, spermatozoa were investigated. Three freezing rates, two semen diluents and three cryoprotectants were tested. Semen frozen in 0.3 mol L^?1 glucose diluent with 10% methanol as a cryoprotectant or in a diluent described by Lahnsteiner with 10%N,N‐dimethylacetamide (DMA) resulted in the highest sperm motility. Fertility was the highest for semen frozen in a glucose–methanol extender but was not significantly different than that for semen frozen in Lahnsteiner's diluent with 10% DMA. Dimethyl sulphoxide (DMSO) at 10% was a relatively ineffective cryoprotectant with either semen diluent. Semen frozen at 6 cm above the surface of liquid nitrogen resulted in a higher post‐thaw sperm motility and fertility than semen frozen at 5 cm. The addition of 7% fresh egg yolk to glucose diluent containing methanol or DMSO did not improve the fertility of frozen‐thawed spermatozoa. However, the addition of 7% fresh egg yolk to glucose–DMA extender significantly improved the fertilization percentages of frozen‐thawed spermatozoa. In conclusion, dilution of semen 1:3 in 0.3 mol L^?1 glucose with 10% methanol and freezing 6 cm above the surface of liquid nitrogen (freezing rate of 40±8°C min^?1, mean±SD from ?5 to ?55°C) is a promising protocol for cryopreservation of Arctic char semen.     

Changes in lipid class and fatty acid composition during development in pike (Esox lucius L) eggs and larvae

To establish the changes which occur during embryogenesis and early larval development, eggs, yolk sac larvae and swim-up larvae of pike were examined for lipid class and fatty acid composition. At a water temperature of 15.5°C, the embryonic phase was short (6 days) and characterized by a 41.3% decline in the lipid content of eggs, accompanied by large reductions in the amount of phosphatidylcholine (41.4% decrease), sterol esters and triacylglycerols (respectively a 41.2% decrease and a 58.1% decrease), but not phosphatidylethanolamine which increased markedly (35.6%). By the time of yolk sac absorption (7 to 11 days after fertilization) the larvae remained inactive and a limited utilization of lipids was observed. Yolk sac phosphatidylcholine was selectively incorporated into larval bodies while the levels of other lipid classes remained unchanged in the yolk. When the swim bladder was filled and the swimming stage was reached (11 days to 13 days af), the yolk was completely depleted and yolk phosphatidylcholine together with yolk triacylglycerols were catabolised. Yolk phosphatidylethanolamine and yolk sterol esters were partly incorporated into the body lipids. In the subsequent swim-up larval stage (13 to 15 days af), a steady decrease in lipids was observed (41.6%). Fluctuations in the levels of polyunsaturated fatty acids, monounsaturated fatty acids or saturated fatty acids examined from eggs or larvae were consistent with changes in lipid classes during pike development. During yolk sac absorption, pike incorporated yolk PUFA released on hydrolysis of phosphatidylcholine into the larval body. The results are discussed with reference to water temperature and in relation to the ontogenic and ecological context of pike development. This revised version was published online in July 2006 with corrections to the Cover Date.     

Dose-Confirmation of Copper Sulfate for Treating Fungus on Channel Catfish,Ictalurus punctatus,Eggs at a Commercial Hatchery

This study at a commercial hatchery was required by the U.S. Food and Drug Administration to provide independent substantiation of the results of previous laboratory dose-confirmation studies on the use of copper sulfate (CuSO₄) to control fungus (Saprolegnia spp.) on the eggs of channel catfish, Ictalurus punctatus. The study compared an untreated control group of eggs to eggs treated with 10 mg/L CuSO₄ in a flow-through system; mean water temperature was 23.5°C. Eggs were treated once daily until the embryos reached the eyed stage (5 treatments). Hatching was complete by day 11, and fry were counted to determine the percentage of survival in each treatment. Fungus was identified by polymerase chain reaction (PCR) as Saprolegnia spp. The mean survival in the control treatments was 4% and 40% in the CuSO₄ treatments; the latter survival was significantly higher, but still lower than normal. This study confirms that 10 mg/L CuSO₄ is an effective treatment to control fungus on catfish eggs when used daily until the eggs are eyed. However, continued treatment of eggs until hatching occurs may be warranted based on fungal growth rates observed after treatments were discontinued.     

Cryopreservation of scaly carp (Cyprinus carpio) sperm: effect of different cryoprotectant concentrations on post-thaw motility, fertilization and hatching success of embryos

The aim of the present study was to determine the effect of various cryoprotectants on post-thaw sperm quality and fertilizing capacity of cryopreserved scaly carp (Cyprinus carpio) semen. The present study focused on freezing of scaly carp sperm utilizing a practical and inexpensive protocol for aquaculture. Semen was diluted with Kurokura’s extender composing 3.6 g/l NaCl, 10 g/l KCl, 0.22 g/l CaCl₂, 0.08 g/l MgCl₂ and 0.2 g/l NaHCO₃. The extender contained three different cryoprotectants (DMSO, DMA and egg yolk) at ratios of 5, 10 and 15 %. Semen was placed into 0.25-ml straws and exposed to liquid nitrogen vapor (?120 °C) using an insulated box with an adjustable tray for 10 min and then plunged into liquid nitrogen (?196 °C) tank. The thawing process was performed in a water bath at 40 °C for 10 s. The results indicated that type of cryoprotectants and their concentrations are rather effective in scaly carp sperm cryopreservation on post-thaw sperm quality, while they are very important in order to obtain high fertilization rates. The highest fertilization rate was determined as 96.4 ± 0.15 % with 15 % egg yolk, while the highest hatching rate was determined as 99.3 ± 0.80 with 15 % DMA. In conclusion, the applied cryopreservation method for scaly carp sperm is suitable to fertilize high amounts of eggs.     

Survival and development of Atlantic salmon eggs and fry at three different temperatures

Groups of Atlantic salmon (Salmo salar) eggs were incubated at 12, 10 and 8° C. At 12° C mortality was high and fry averaged little more than half the weight of those hatched at 10 or 8° C. Development of alevins to the ‘swim-up’ stage was also abnormal at 12° C. The results suggest that 10° C is optimal for incubating Atlantic salmon eggs. For the period between hatching and swim-up, the most favourable temperature varies according to the temperature used during incubation. Mortality rate during the first 6 weeks of feeding was correlated with mortality during earlier development. Total numbers of day-degrees required by the eggs and fry to reach the eyed, hatching, and swim-up stages of development were significantly less at 12° C than at 10 or 8° C. However, total day-degrees required to reach an average weight of 0.5 or 0.6 g were almost the same regardless of temperature during hatching.     

Short-term storage and cryopreservation of milt from Atlantic salmon and sea trout

Experiments on short-term preservation of sperm were performed with Atlantic salmon (Salmo salar). Fertility was maintained for up to 10 days when 2 mm thick samples were stored at 0° C under an oxygen atmosphere in the presence of antibiotics (125 IU penicillin and 125 μg streptomycin per ml sperm). Fertility was completely lost after 24 days. Sperm stored without antibiotics fertilized 100% of eggs after 6 days.

Cryopreservation was carried out with milt from Atlantic salmon and sea trout (Salmo trutta). Semen mixed with extender was frozen on dry ice (pellets) with subsequent storage in liquid nitrogen. Sperm pellets were thawed in a 0.12-M NaHCO₃-solution (10° C) before insemination. The suitability of an extender as previously described by Stoss and Holtz and of a 0.3-M glucose solution with the addition of 10% DMSO, was tested on two different batches of sperm and eggs in Atlantic salmon and sea trout. In addition, the extender earlier reported by Mounib and an aqueous solution of 10% DMSO were only used in Atlantic salmon with one batch of gametes.

Insemination with cryopreserved Atlantic salmon sperm resulted in 36 to 91.3% eyed eggs (control = 100). The differences were caused by the type of extender and the batch of gametes employed. The very simple extender consisting of 0.3 M glucose and 10% DMSO only was the most successful. Results with cryopreserved sea trout sperm ranged between 38.6–54.8% eyed eggs, showing no difference between treatments.     

Effect of Extenders and Osmotic Pressure on Storage of Eggs of Ornamental Common Carp Cyprinus carpio at Ambient and Refrigerated Temperatures

The eggs of ornamental (koi) common carp Cyprinus carpi0 were stored at ambient temperature (～22–25 C) and at refrigerated temperatures (0–20 C) in extenders at different osmolalities. The treatments evaluated were dry (control), calcium‐free Hanks’balanced salt solution (C‐F HBSS), salt (NaCl), synthetic ovarian fluid (SOF), and Kurokura #2 (K2). In the first study, eggs were placed in extenders at osmolalities ranging from 130 to 450 mOsmol/kg and were fertilized after 2 h. The percentage of eyed embryos (our measure of fertilization capacity) was calculated 24 h later, and percent hatching was calculated at 60 h. Fertilization capacity of eggs suspended in C‐F HBSS (28%) or SOF (37%) was highest (P= 0.0001) at 250 mOsmol/kg, while eggs stored dry (control) had a fertilization capacity of 24%. Fertilization capacity of eggs suspended in NaCl (40%) or K2 (39%) was highest (P= 0.0001) at 200 mOsmol/kg. The percent of eyed embryos and percent hatch were found to be positively correlated (r= 0.9914). In the second study, eggs were stored in these extenders with the most effective osmolality from the previous study to evaluate percent eyed embryos and hatching over time. Samples of eggs were fertilized at every hour for 7 h. Eggs in the extenders C‐F HBSS and SOF yielded the highest (P= 0.0001) percent eyed embryos during 7 h. Percent hatch of these eggs was not significantly different (P= 0.1258) among treatments at each time interval. Eggs stored in the extenders SOF, C‐F HBSS, and NaCl had higher fertwzation capacity (P= 0.0271) at 7 h than did the dry control. Eggs were also stored at refigerated temperatures in these four extenders at the most effective osmolalities from the first study. A dry control (no extender) was also compared. The third study compared quality of eggs stored for 0, 2, 4, or 6 h in each of the extenders at 5 C or at ambient temperature (～22–25 C). Eggs suspended in C‐F HBSS had significantly higher fertilization capacity at ambient temperature over time than did eggs stored in NaCl, SOF, K2, or the dry control. Eggs suspended in C‐F HBSS and the dry control had significantly higher fertilization capacity at 5 C over time than did eggs stored in NaCl, SOF, or K2. Eggs held dry had higher hatch at ambient temperatures (P= 0.0001) and at 5 C (P = 0.0002) over time than did eggs stored in any extender. At 6 h, fertlllvltion capacity with eggs in C‐F HBSS or K2 was higher than with NaCl, SOF, or the dry control. The fourth study used C‐F HBSS (250 mOsmol/kg) as the extender to evaluate fertilizing and hatching ability during storage at temperatures 0, 5, 10, 15, 20, or 25 C. Eggs were fertilized after 0, 1, 3, 6, 9, or 12 h of storage. Eggs stored at 15 C had significantly higher fertilization capacity (P= 0.0001) than at any other temperature. Eggs stored at 15 C and 10 C had significantly higher hatch (P= 0.0001) than at any other temperature. Fertilization capacity at 12 h was significantly higher in eggs stored at 10 C (33%) or 15 C (29%) than at any other temperature. Storage of koi carp eggs in C‐F HBSS at remgerated temperatures extended fertilizing ability for as long as 12 h compared to storage in NaCl, SOF, K2, or the dry control.     

Comparative efficacy of Pyceze® (bronopol) in controlling mortality of brown trout Salmo trutta eggs

The efficacy of Pyceze^® (Novartis Animal Vaccines) and Proxitane^® 0510 (Solvay Interox) in controlling the mortality of eggs was studied in brown trout Salmo trutta eggs under the usual incubation conditions in a hatchery affected by saprolegniosis. Eggs from eight spawnings and from two lines of brown trout were used. The cumulative mortality of eggs at the start of the eyed stage (M₁) and at hatching (M₂) was measured, as was the percentage of eggs with fungal infection at weekly intervals during the green stage. Mortality at M₂ with Pyceze^® ranged between 2.38% and 12.61% depending on the batch, with a mean of 6.10%. Mortality at M₂ with Proxitane^® varied between 5.83% and 43.86%, with a mean of 22.36%. Fungal colonization at the start of the eyed stage ranged between 0% and 0.15% when Pyceze^® was used and between 0.82% and 12.50% with Proxitane^®. Mortality rates were higher among those eggs left untreated. The results indicate that Pyceze^® (bronopol) is efficacious in controlling mortality caused by Saprolegnia spp. and other biological factors in fertilized brown trout eggs, as has been demonstrated previously in other salmonid species.     

Patchiness of eggs, larvae and juveniles of European hake Merluccius merluccius from the NW Mediterranean

We estimated patchiness for different life stages (egg, larvae and juveniles) of European hake (Merluccius merluccius) from two ichthyoplankton surveys (September and November 1999) and four bottom‐trawl surveys (November 1998, February, June and September 1999). Lloyd's patchiness P = 1 + 1/k, where k is the dispersion parameter of the negative binomial distribution, was calculated. Juveniles showed lower indices of patchiness than early ontogenetic stages. In juveniles, patchiness was highest for smaller size classes (3.5–5 cm TL) settling to their near‐bottom habitat and was very low for those larger than 5 cm up to the 30–35 cm size class (approximately 3 yr of age, 50% mature individuals). Patchiness of settling juveniles was especially high in the June and September surveys, coinciding with peak recruitment to the bottom. Both eggs and yolk sac larvae showed patchiness values <10, increasing in flexion stage larvae from 12 to 20. This pattern is different to that observed in other studies for pelagic species, where patchiness of early‐stage eggs is very high, but coincides with results obtained for other demersal species with pelagic eggs. Factors that may explain these patterns are discussed in light of oceanographic conditions, adult distribution and larval ontogenesis.     

Spawning behaviour,early development and first feeding of the bluestriped angelfish [Chaetodontoplus septentrionalis (Temminck & Schlegel, 1844)] in captivity

Successful natural spawning of Chaetodontoplus septentrionalis in captivity from 19 March to 11 May, 2008 is described for the first time. A single male dominates a harem of two females, spawning with each at dusk, from 10 min before to 20 min after sunset. Each female laid an average 119 × 10³ eggs during the spawning period. Fertilized eggs were spherical, buoyant and had a diameter of 0.83 ± 0.02 mm (mean ± SD). Embryonic development lasted 15–18 h at 28.1 °C. Newly hatched larvae were 1.60 ± 0.07 mm in total length (TL) with 27 myomeres. Larvae completed yolk absorption within 3 days post hatching (ph) at 3.01 ± 0.08 mm TL. Ten days ph, the larvae had attained 3.95 ± 0.12 mm TL. Larvae were fed either 100% s‐type rotifers (Brachionus rotundiformis), 100% copepods (Microsetella sp.), a combination of the two (50%:50%) or without live feed (starved control) to determine the effect of live feed on the survival rate. The survival was significantly (P<0.001) higher in larvae fed a combination of diet than the others. These results indicate that C. septentrionalis is a potential species for captive breeding programs and the use of a combination of diet (s‐type rotifers and copepods) may be a suitable first food for the larvae.     

First results on a relation between ovarian fluid and egg proteins of Salmo trutta and egg quality

By use of sodium dodecyl sulfate polyacrylamide gel electrophoresis, ovarian fluid proteins and main proteins of unfertilized eggs were qualitatively and quantitatively investigated in the brown trout, Salmo trutta, to see whether some of them were correlated with the rate of embryos reaching the eyed embryo stage. In the ovarian fluid, 12 types of proteins in the range of 39–166 kDa were detected whereby three proteins were lipoproteins and two were glycoproteins. Ovarian fluid proteins with a molecular weight of 85, 68, 62 and 39 kDa were negatively correlated with the percentage of eyed stage embryos. The statistical significance of the relations was low in simple and multiple regression models (R²≤0.534) indicating that the relations were influenced and superposed by other factors. Therefore, ovarian fluid proteins give only poor information about maturity and quality of eggs. In the eggs, nine major types of proteins in the range of 95–15 kDa were identified. The 95 kDa protein was a lipoprotein, the 85 and the 62 kDa protein were glycoproteins, and the 15 kDa protein was a phosphoprotein. The 95, 85, 77 and 39 kDa protein were positively correlated with embryo survival to the eyed embryo stage. The explanatory effect of the multiple regression model was very high (R²=0.961) indicating that distinct egg proteins are closely related with egg quality.     

Embryonic development and morphology of eggs and newly hatched larvae of Pacific herring <Emphasis Type="Italic">Clupea pallasii</Emphasis>

The embryonic development and morphology of eggs and newly hatched larvae of the Pacific herring Clupea pallasii were described using laboratory-reared specimens originating from the Miyako Bay stock. The eggs were almost spherical in shape, 1.33–1.46 mm (mean: 1.38 mm) in diameter, and had a thick adherent chorion. They had a segmented pale yellow yolk, no oil globule, and a relatively wide perivitelline space. A subgerminal cavity was observed during the gastrula period, whereas the blastocoel did not appear. Mass hatching occurred by 271 h 45 min after fertilization, and the newly hatched larvae were 7.1–7.7 mm (mean: 7.5 mm) in total length with 53–56 myomeres at 9.6°C. The embryonic development of Pacific herring was substantially similar to that of zebrafish Danio rerio, American shad Alosa sapidissima, as well as Atlantic herring Clupea harengus, and generally followed the basic developmental pattern of teleosts. However, Pacific herring larvae hatched at a more developed stage than some other clupeoids, such as Japanese sardine Sardinops melanostictus, and the progressed developmental stage at hatching could be interpreted as an advanced adaptation.     

Computer-controlled freezing of rainbow trout Oncorhynchus mykiss (Walbaum) spermatozoa for routine programmes

The effects of extender composition and freezing rate on cryopreservation efficiency of refrigerated spermatozoa of rainbow trout Oncorhynchus mykiss (Walbaum) were evaluated in order to test the suitability of a computer-controlled ultrafreezer to cryopreserve milt samples obtained in field conditions and stored for several hours. A very highly significant first-order interaction between freezing rate and the type of extender was found. Six of the eight experimental variants did not differ significantly, resulting, after fertilization of eggs with cryopreserved sperm, in a range of 62.3–74.8% of eyed embryos. This procedure was effective for samples stored at 1 °C for 2 days.     

Effects of incubation water hardness and salinity on egg hatch and fry survival of Nile tilapia Oreochromis niloticus (Linnaeus)

This study examined the effects of water hardness and salinity on yolk sac larvae and swim‐up fry survival of Nile tilapia, Oreochromis niloticus (Chitralada strain), eggs during artificial incubation. Four experiments were conducted to evaluate the effects of hardness, salinity and the sources of saline incubation water. High water hardness treatments (500–4200 mg L^?1 as CaCO₃) resulted in higher yolk sac larvae and swim‐up fry survival than low water hardness treatments (50.0 and 132 mg L^?1 as CaCO₃); although yolk sac larvae and swim‐up fry survival did not differ among the high or low hardness treatments. Salinity of 4.0 g L^?1 using seawater, and 4.0 and 8.0 g L^?1 using unprocessed common salt resulted in the higher survival rate of yolk sac larvae and swim‐up fry than other salinity treatments. Yolk sac larvae and swim‐up fry survival was found to decrease with the increase in salinity and increase with the increase in water hardness. The present study demonstrated the positive effects of increased water hardness level (>132 mg L^?1) on yolk sac larvae and swim‐up fry survival. The study also showed that seawater salinity of 4 g L^?1 was the most appropriate salinity level for incubating Nile tilapia eggs.     

Cryogenic and Refrigerated Storage of Rainbow Smelt Osmerus mordax Spermatozoa

The effects of extender composition, cryoprotectant, and freezing rate on post-thaw rainbow smelt Osmerus mordax sperm motility were examined, and the fertilization capacity of fresh and post-thaw sperm were compared. The highest post-thaw motility (75%) was obtained when milt was diluted 1:3 with an extender containing 600 mM sucrose supplemented with 10% dimethyl sulfoxide and 1.5% bovine serum albumin and frozen at a rate of –20 C/min. Post-thaw motility for sperm stored in this extender was similar to fresh sperm and did not change after 90 d of storage. Furthermore, there were no differences in fertilization rate or embryo survival to the eyed stage between fresh and post-thaw sperm frozen in this extender. The lowest post-thaw motility was observed when sperm were frozen with methanol at a rate of -30 C/min. Refrigerated sperm diluted 1:3 with the 600 mM sucrose extender remained motile for 30 d. These data demonstrate that rainbow smelt spermatozoa can be effectively used following short and long-term storage using a simple, sucrose-based extender.     

斑点鳟仔、稚、幼鱼的形态发育

以斑点鳟(Oncorhynchus mykiss)发眼卵为材料,进行了发眼卵的人工孵化及仔鱼的人工培育研究。对斑点鳟仔稚鱼的形态发育进行了系统观察,描述了各发育期的形态特征,并测定生长参数。斑点鳟早期生活史的分期如下:初孵仔鱼全长14.25±0.45 mm,体重85±5 mg,肛门未开口,营内源性营养,脊柱末端向上弯曲;10 dph开始上浮,投喂卤虫;12 dph开口摄食,开始营内外源混合性营养;14 dph鳍膜消失,各鳍独立;16dph开始投喂配合饵料;24 dph卵黄囊吸收完毕,营完全外源性营养;34 dph,体侧形成8～9个幼鲑斑;44 dph各鳍的鳍条发育健全;60 dph幼鱼外形为纺锤形,与成鱼相同。研究表明,斑点鳟早期生长发育过程中全长、肛门前长、水平眼径和头高的生长情况基本相同,0～16 dph生长速度较快,16～44 dph生长速度较慢,44 dph后生长速度再次变快。     

Additive effects of advanced temperature and photoperiod regimes and LHRHa injection on ovulation in Atlantic salmon (Salmo salar)

In order to evaluate manipulation of spawning time as a potential means to extend 0+ Atlantic salmon (Salmo salar) smolt production in Tasmania, Australia, female salmon were exposed to a natural/simulated natural (42°S) photoperiod or an advanced (L:D 9:15) photoperiod from the austral summer solstice (20 December) under natural or advanced (~ 6 °C below natural temperature) temperature conditions. In late summer (26 February) injections of a commercial LHRHa preparation or vehicle (propylene glycol) commenced. Regular ovulation checks were conducted and ova were fertilised using milt from LHRHa-injected males held under matching photo-thermal conditions. Plasma levels of 17β-estradiol (E₂) and testosterone (T) were monitored and reproductive success (cumulative % ovulation, % fertilisation and % survival to the eyed-egg stage) was recorded. Ovulations commenced first (09 March) in LHRHa-treated fish that experienced advanced photoperiod and thermal regimes whereas sham-treated fish exposed to natural photoperiod and temperature conditions where the last to ovulate (22 May-08 June). Treatment-related sequential changes in the timing of ovulations were reflected by sequential advances in the timing of peaks in plasma levels of E₂ and T. The fertilisation of ova from LHRHa-treated fish that experienced advanced photoperiod and thermal regimes was significantly reduced (~ 52%) relative to all other treatments (> 80%) but there were no significant treatment-related differences in the survivals of eggs to the eyed stage (~ 50-90%). Consequently, a maximum advance in the timing of median ovulation of 71 days and commercially acceptable eyed-egg yields were generated, demonstrating that combinations of photoperiod, thermal and hormone treatments may be employed to significantly extend spawning and thereafter increase the availability of 0+ smolts for grow-out.