Induction of diploid gynogenesis in turbot <Emphasis Type="Italic">Scophthalmus maximus</Emphasis> with left-eyed flounder <Emphasis Type="Italic">Paralichthys olivaceus</Emphasis> sperm

Turbot Scophthalmus maximus exhibits sexually dimorphic growth, with females growing faster and reaching larger adult sizes than males. Thus, development of techniques for preferentially producing females is necessary to optimize production of these species. In this paper, gynogenetic diploids of turbot were induced by activating egg development with ultraviolet (UV)-irradiated left-eyed flounder Paralichthys olivaceus sperm combined with cold shock to prevent extrusion of the second polar body. The results of UV irradiation experiments showed that survival, motility, and duration of activity of P. olivaceus sperm generally decreased with increase in UV dose. The typical Hertwig’s effect was observed after fertilized turbot eggs with UV-irradiated P. olivaceus sperm and the optimal UV dose for gynogenetic haploid production was 36,000 erg mm⁻². At 15°C, appropriate timing of cold shock for retention of the second polar body in turbot eggs was at 6 min after fertilization. Results of different combinations of two shock temperatures (1 or 3°C) and four shock durations (15, 25, 35 or 45 min) at 6 min after fertilization demonstrated that shock of 25 min at 1°C gave the highest production of diploid gynogens (39.58% relative to its diploid control). The results of this study reveal that the use of UV-irradiated P. olivaceus sperm for activation of turbot eggs and cold shock for polar body retention is an effective method to produce gynogenetic offspring.     

Meiotic Gynogenesis Induction in Muskellunge Esox masquinongy by Thermal and Pressure Shocks

Mass production of fast‐growing, all‐female muskellunge Esox rnasquinongy by gynogenesis requires optimized techniques of preventing second polar body extrusion. Heat, cold, and pressure shocks were evaluated for their efficiency of doubling the maternal genome. Muskellunge eggs (20–40 g) were activated with 1 mL ultraviolet (UV)‐irradiated (1,248 J/ m²) heterologous sperm of yellow perch Perca flavescens. Survival and ploidy (by flow cytometry) were determined during the eyed‐stage. Cold shocks of 1.3 × 1 C were applied at 5 or 20 min after gamete activation with water (time of initiation, TI) for a duration of 150 min and pressure shocks of 48,263 or 55,158 kPa (7,000 or 8,000 psi, respectively) at a TI of 4 rnin for 12 min. These shock treatments resulted in 43.7–95.0% diploid gynogens with corresponding yield of diploid gynogen (percent diploid gynogens × total percent survival) of 2.6–11.I%. Cold shocks applied at TI of 5 or 20 min after activation resulted in statistically similar percent survival, percent diploid gynogens, and yield of diploid gynogens. Heat shocks of 31 × 0.1C applied at a TI of 5 to 15 min for a duration of 5 min resulted in 4.8–21.1% diploid gynogens with yields of 0.1–0.4%. Cold and pressure shocks have better potential than heat shock for preventing the second polar body extrusion. Muskellunge eggs activated with UV‐irradiated yellow perch sperm, but not exposed to shock, resulted in 100% haploids with survival of 2.3–5.8%. No viable embryos were produced from the hybrid cross between muskellunge and yellow perch, thus, all diploids produced after the shock treatments were unambiguous meiotic gynogens. Muskellunge eggs fertilized with fresh muskellunge sperm (controls) showed 60.4–64.0% survival to the eyedstage and 100% diploidy. Considering that the sex‐determining mechanism in muskellunge follows the WZ female, ZZ male system, future efforts should be directed to test the efficiency of cold and pressure shocks for mass‐producing gynogenetic super female (WW) muskellunge.     

Induction of meiotic gynogenesis in the stinging catfish Heteropneustes fossilis (Bloch) and evidence for female homogamety

This study reports the results on induced meiotic diploid gynogenesis and female homogametic nature in the Indian catfish, Heteropneustes fossilis. The eggs of H. fossilis were inseminated with conspecific sperm. The sperm suspension was diluted to 1 × 10⁷ sperm mL⁻¹ in Hanks balanced salt solution. Sperm were irradiated under UV light, with the exposure time ranging from 15 to 360 s (7500 ergs mm⁻² for 60 s). The genetic inactivation of paternal chromosomes was confirmed by chromosome counting from the larval cells and the larvae also had a characteristic haploid syndrome. A typical ‘Hertwig effect’ in the yield of hatched larvae was observed with doses of UV exposure >75 s (9375 ergs mm²). Larvae resulting from sperm UV irradiated above 120 s (15 000 ergs mm²) were 100% haploids. Application of heat shock to the activated eggs was effective in suppressing the release of the second polar body (meiotic gynogenesis) and resulted in diploid gynogenetic larvae morphologically identical to those of the control. The best yield of diploid gynogens (49.3% with respect to the control) was found to be at 6 min after egg activation and the heat shock at 41 °C for a 1-min duration, at an ambient water temperature of 27 °C. A total of 113 diploid gynogenetic fry from seven different female fish were reared and subjected to sexing. All gynogenetic fish were female in contrast to the control, which had a mean sex ratio of 56.7% females (which was not significantly different from 50% female). From these results, the sex determination mechanism in H. fossilis was presumed to be female homogamety.     

Artificial gynogenesis and assessment of homozygosity in meiotic gynogens of spotted halibut (<Emphasis Type="Italic">Verasper variegatus</Emphasis>)

In the present study, methodology of gynogenetic induction in spotted halibut were developed and optimized; the sex ratio of putative meiotic gynogenetic diploids was determined using AFLP-based molecular sexing technique; the homozygosity of gynogenetic population was assessed as opposed to cultivated population. The results showed that high percentage of meiotic gynogenetic diploids were generated when the eggs fertilized with irradiated heterologous sea perch frozen sperm (30–50 mJ cm⁻²) were cold shocked in sea water of −1°C for 40–75 min at 5 min after fertilization. About 15,200 diploid gynogenetic larvae were achieved and they exhibited normal morphology similar to diploid control. The gynogenetic diploids were 100% female, which first confirmed the female homogamete (XX/XY) sex determination in spotted halibut. The genetic analysis showed that the average H _O was, respectively, 0.404 and 0.724 in gynogenetic population and cultivated population, indicating an increase of homozygosity in gynogenetic population.     

Chromosome set manipulations in the black carp

The mollusc-eating black carp (Mylopharyngodon piceus) has economic and health-care potential for biological control of nuisance aquatic molluscs. The present study investigates the production of gynogenetic-monosex and triploid-sterile populations of black carp. The goal was to provide a method which would eliminate unwanted biological and environmental impacts of introducing this exotic species into areas with nuisance mollusc infestation. Meiotic gynogenesis was induced by inseminating black carp eggs with UV-irradiated (800 Jm⁻²) sperm of common carp (Cyprinus carpio) or Japanese ornamental (koi) carp. Diploidy was restored through retention of the second polar body (2PB), by shocking activated eggs at 1–8 min post-fertilization (embryological age of 0.07–0.57τ₀, a parameter defined by the cell cycle duration) at 1 min intervals, with heat-shocks (41.0±1.0 °C for 1 min) or pressure-shocks (7500–7600 psi for 1.5 min). Highest survival was found when embryos were heat-shocked 1.5–4.5 min post-fertilization (0.10–0.25τ₀). The highest survival of free-swimming larvae from pressure-shocked eggs, was achieved at 7500 psi at 1–2 min post-fertilization (0.08–0.16τ₀). Triploidy was induced by retention of 2PB following normal fertilization. Batches of 30 000 eggs were fertilized with intact sperm and pressure-shocked (6000–8500 psi for 1.5 min) 2 min post-fertilization (0.15–0.16τ₀). The highest survival of triploid swim-up larvae was 5.1% in eggs shocked with 7500 psi. In random samples of individual larvae taken from each treatment, triploidy was analysed by cytofluorometry of the cellular DNA content. In DNA analysis performed in fingerlings (N≥15), 50% of the fish were triploids.     

Insemination, fertilization and gamete management in tench, Tinca tinca (L.)

Various procedures for artificial insemination in tench, Tinca tinca (L.) were re-examined with evaluation of fecundity of males and females among different tench strains. The objectives of this study were to enhance fertilization and hatching rates through optimization of the activation solution, the insemination process, the activation of gametes, and the elimination of eggs stickiness. Sperm for all experiments was collected directly into immobilization solution of modified Kurokura solution containing 180 mM of NaCl and stored at 2 °C for 2.5–5 h prior to the experiment. When dechlorinated tap water was used for activation a gamete ratio of 1150 spermatozoa per egg showed the best significant fertilisation and hatching rates. Optimal ratio between eggs (weight in g) and activation solution (in cm³) was 1:1. Different concentrations of activation solutions such as NaCl from 0 to 68 mM (0–136 mOsmol kg⁻¹) without buffer statistically decreased fertilization and hatching rates. The activation solution containing 17 mM of NaCl, 10 mM Tris–HCl, pH 8 and 9 significantly increased fertilization and hatching rates compared to dechlorinated tap water of pH 7 or activation solution containing 17 mM of NaCl, 10 mM Tris–HCl, pH 6 and 7. Adhesiveness of the eggs was successfully removed by incubation in Alcalase and activity: 3.16 Anson units per cm³.     

Effect of temperature on the development of eggs and the daily pattern of spawning of round herring <Emphasis Type="Italic">Etrumeus teres</Emphasis>

Effect of temperature on the development of eggs of round herring Etrumeus teres was experimentally examined to construct a temperature-dependent egg development model. Mature fish were collected in the field and their eggs were artificially fertilized onboard. The eggs were incubated at nine temperatures set between 14.0 and 25.0°C. All eggs at the lowest three temperatures, 14.0°C, 15.0°C, and 16.0°C, ceased development and died at various stages before hatching. Durations required to hatching after fertilization ranged from 38.0 h at 25.0°C to 90.0 h at 17.5°C. The temperature-dependent egg development model, i.e., egg age in hours (y _i,t ) at the ith stage and temperature t (°C), was expressed as: y _i,t  = 4.604 × exp(−0.100 × t −0.129 × i) × i ^2.593. From the application of the model to early-stage eggs collected in the field, it is concluded that round herring starts spawning immediately after sunset and almost completes spawning by midnight. The temperature-dependent egg development model and the daily pattern of spawning presented in this study are essential tools for developing the daily egg production method to estimate the spawning stock biomass.     

Pre-spawning water temperature affects sperm respiration and reactivation parameters in male carps

Concentration, ability to motility, motility during the second activation (reactivation), and endogenous respiration were studied in sperm from two experimental groups of carp males. Group 1 was maintained for 7 days at 15°C (cold water (CW) group), whereas the second group was subjected to a temperature of 20°C (warm water (WW) group) before sperm sampling. Reactivation were achieved after incubation of firstly activated sperm in media with osmotic pressure adjusted up to 300 mOsm*kg⁻¹ by increasing K⁺ concentration. Statistically significant reduction of spermatozoa concentration in CW samples versus WW (from 46.0 ± 12.5 (15°C) to 59.3 ± 7 10⁹ (20°C) spermatozoa /ml) have been observed. The sperm of the CW group required a significantly longer incubation time (37 min) under isotonic conditions to achieve a maximum percentage of potent motility at repeated activation than the WW group (23 min). After activation of sperm motility, an increase of respiration rate up to maximum level has been found, this level remained the same under condition of recovering the potential to repeated activation. During the sperm movement respiration rate, in CW group (6.1 nmolO₂/min/10⁹spermatozoa) and WW (3.9 nmolO₂/min/10⁹spermatozoa), was significant higher compared to nonactivated sperm (2.4 nmolO₂/min/10⁹spermatozoa for CW and 1.1 nmolO₂ /min/10⁹spermatozoa for WW). And keeping males for 7 days at 15°C increase the respiration rate of sperm.     

Induction of triploidy in large yellow crocker Pseudosciaena crocea (Richardson, 1846): effects of pressure shocks and growth performance in the first rearing year

The precociously sexual maturation in large yellow crocker Pseudosciaena crocea has become a serious problem. In an attempt to solve this problem, the production of sterile triploids could be an effective strategy. In this study, triploid P. crocea was obtained by subjecting fertilized eggs to pressure shock. Flow‐cytometry analysis was used to assess ploidy level. In terms of triploid rate and hatching rate, the optimal conditions of pressure shock for triploidy induction in P. crocea were 7500 psi for 3 min shock at 3 min after fertilization at 20 °C. With the application of these parameters, 100% triploid fish were produced. During the first rearing year, triploid P. crocea had a similar growth performance compared with its diploid counterpart before the age of 8 months and showed a significant advantage at the age of 10 and 12 months in body weight and body length (P<0.05). At the age of 12 months, the carcass weight of triploids was markedly higher than that of diploid control, and gonadal somatic index was significantly lower than that of their diploid control. During the first rearing year, survival in triploid group was 76.44%, inferior to its diploid control (83.21%).     

Motility Parameters of Rainbow Trout (<Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis>) Spermatozoa in Relation to Sequential Collection of Milt,Time of <Emphasis Type="Italic">Post-mortem</Emphasis> Storage and Anesthesia

The present study investigated the effects of sequential collection of milt, time of post-mortem storage and anesthesia on rainbow trout (Oncorhynchus mykiss) sperm motility parameters (using computer-assisted sperm analysis – CASA) as well as seminal plasma osmolality and sperm concentration. The post-mortem storage and time of anesthesia altered motility characteristics of rainbow trout sperm to different extents. The moderate impact of time of anesthesia was manifested in a shortened duration of sperm motility after 10 min exposure of fish to anesthetic. The prolonged post-mortem storage (≥40–60 min), in addition to lowering sperm motility duration, also significantly influenced sperm motility parameters, such as sperm velocities, percentage of motile sperm and sperm trajectory parameters. These results clearly demonstrate that when milt from sacrificed fish is used for sperm motility studies, the time of post-mortem storage significantly alters sperm motility characteristics. Since sperm motility rate and swimming velocity could predict fertilizing ability, detrimental effects of prolonged post-mortem storage may lead to reduced fertilization success. Sperm concentration and seminal plasma osmolality were lower in the first fractions and increased with successive collections of milt. It suggests the presence of urine contamination of the first milt fractions which were collected by stripping. Therefore, testing of sperm concentration and/or seminal plasma osmolality should be mandatory while handling stored milt.     

Optimisation of larval culture of the mussel <Emphasis Type="Italic">Mytilus</Emphasis><Emphasis Type="Italic">edulis</Emphasis> (L.)

The blue mussel Mytilus edulis is a commercially important species whose fishery and culture generally relies on natural spat collection. Hatchery-production could provide an alternative source of seed, enabling reliable expansion of the industry. Mussel spawning and larval rearing trials were carried out to optimise elements of hatchery production. Culturing fertilised eggs at low density (20–200 eggs cm⁻²) rather than high density (400–720 eggs cm⁻²) significantly improved the quality of first veliger larvae and differences in this improvement were evident between the eggs from different females (maternal effects). Veliger larval growth at 17 or 21°C was significantly faster than growth at 14°C. Feeding veliger larvae an identical total ration either daily or at 2–3 day intervals did not significantly affect their growth. Different microalgal diets (1: Isochrysis sp. (clone T-ISO), 2: Chaetoceros calcitrans forma pumilus, 3: C. muelleri, 4: mixed Isochrysis sp. (clone T-ISO) and C. calcitrans f. pumilus, and 5: mixed Isochrysis sp. (clone T-ISO) and C. muelleri) were tested on veliger larval growth and mixed diets outperformed single-species diets.     

Effect of extenders on sperm cryopreservation of African catfish, Clarias gariepinus (Burchell)

The effect of extenders was studied on the cryopreservation of sperm from African catfish, Clarias gariepinus (Burchell). The following six basic extenders were tested: fructose, glucose, sucrose, NaCl, KCl solutions and the artificial seminal plasma of the African catfish. Each of these extenders was tested both with and without buffer systems (i.e. NaHCO₃-CO₂ and Tris-HCl) by using 10% dimethyl-sulphoxide (DMSO) as a cryoprotectant. The two-step freezing was carried out in a programmable freezer by using the following freezing rates: (1) 4 °C min^–1 between 3 and –4 °C; (2) and 11 °C min^–1 between –4 and –80 °C. The best post-thaw motility (25%) was achieved with 333 mmol L^–1 fructose solution and NaHCO₃ buffer. The fertilization experiments were carried out with unbuffered fructose and glucose extenders using various amounts of sperm and two fertilization methods: (1) dry and (2) wet. The best fertilization rates were achieved with 75 μL of sperm and wet fertilization with glucose extender, or 100 μL of sperm and dry fertilization in case of fructose – both methods fertilized 96% of all eggs.     

Fertilization by refrigerator stored sperm of the Indian major carp, Labeo calbasu (Hamilton, 1822)

This study reports on the spermatological properties, and on the development of a protocol for refrigerator storage (4°C) of Labeo calbasu (Hamilton, 1822) sperm for artificial breeding. Volume, motility, concentration and pH of the freshly collected sperm were 2.21 ± 0.53 (μL g^?1 of fish weight) (mean ± SD), 95 ± 1 (%), 1.93 ± 0.44 × 10⁹ (cells mL^?1) and 7.56 ± 0.17 respectively. Sperm activation was evaluated at different osmolalities of NaCl solution, and motility ceased completely when osmolality of the extender was ≥287 mOsmol kg^?1. Sperm retained motility for 24, 72 and 108 h, after refrigerator storage when sperm were undiluted, suspended in Alsever's solution and suspended in Alsever's solution containing 5% methanol respectively. Fertilization rate of fresh eggs with sperm stored at 4°C in Alsever's solution and Alsever's solution containing 5% methanol was 77% and 60% with a hatching rate of 60% and 43% respectively. The fertilization and hatching success of the stored sperm suggests potential to use refrigeration for transporting genetic material to hatcheries for artificial breeding of L. calbasu in Bangladesh.     

Induction of Diploid Androgenesis in the Stinging Catfish,Heteropneustes fossilis

This study reports the results on induced diploid androgenesis in the Indian catfish, Heteropneustes fossilis. Ultraviolet (UV) irradiation was used to inactivate maternal genome of H. fossilis. Complete inactivation of maternal genome was recorded at 12,500 ergs/mm². These genome‐inactivated eggs of H. fossilis were inseminated with conspecific sperm. The sperm suspension was diluted to 1 × 10⁷ sperm ml/L in Hank's Balanced Salt Solution. Egg viability was assessed for different exposure durations at fertilization, hatching, haploidy, and diploidization. Majority of the larvae derived from irradiated eggs had abnormal appearance. Complete inactivation of maternal genome was detected by haploid syndrome and confirmed by chromosome counting (n = 29). These eggs activated with sperm were subjected to heat shock at 40 and 41 C for different postactivation times and durations. Diploid androgens had a normal appearance as controls and confirmed by chromosome counting (n = 58). A maximum of 21 and 14% of diploidization was recorded at 30 min after activation, at 40 and 41 C, which corresponds to the first cleavage suppression time.     

Effects of cooling rate on post-thaw motility and fertility of Japanese pearl oyster <Emphasis Type="Italic">Pinctada fucata martensii</Emphasis> spermatozoa

The post-thaw motility and fertility of Japanese pearl oyster sperm show large variances, even among sperm samples obtained from the same individuals. This study aimed to clarify the factors that cause such differences. Spermatozoa were diluted 50 times with diluent comprising 10 % methanol, 18 % fetal bovine serum, and 72 % seawater, and dispensed into 0.25 ml straws. A total of 59 straws were cooled, one by one, at 11 different heights from the surface of liquid nitrogen (LN) to −50 °C, and then immediately immersed in LN. After thawing the straws, the relationships between the cooling rate and the post-thaw motility and post-thaw fertility of the spermatozoa were examined. Both the post-thaw motility and the post-thaw fertility showed a sharp peak when the straws were cooled at around −20 °C/min. There was a strong correlation between post-thaw motility and fertility (P < 0.001). There was a large difference in the cooling rates and the post-thaw motilities and fertilities of the spermatozoa, even between straws cooled at the same height. These results indicate that the optimum range for the cooling rate of oyster spermatozoa is quite narrow, and the method of cooling straws at a fixed distance from the LN surface is unsuitable for the cryopreservation of Japanese pearl oyster spermatozoa.     

Cryopreservation of filefish (Thamnaconus septentrionalis Gunther, 1877) sperm

The present study examined the possibility of long‐term storage, by cryopreservation in liquid nitrogen, of the sperm of filefish (Thamnaconus septentrionalis). Changes in motility, survival rate, ultrastructure and fertilization rate of the sperm after freezing and thawing were tested. For selection of the immobilizing solution, artificial seawater (ASW) of 250, 350 and 450 mOsmol kg^?1 were tested. Sperm motility was significantly inhibited in 350 mOsmol kg^?1 ASW, and restored entirely after 100% ASW (1200 mOsmol kg^?1) was added. Two cryoprotectants, dimethyl sulphoxide and glycerol, were employed. The sperm was diluted at the ratio of 1:6 with the extenders, and frozen at a freezing rate of ?40°C min^?1 to ?100°C after equilibration for 10 min at room temperature, followed by plunging into liquid nitrogen. The highest post‐thawed sperm motility and survival rate were obtained with 5% glycerol. Afterwards, the effect of different freezing rates was examined using 5% glycerol as a cryoprotectant, and the rate of ?30°C min^?1 to ?100°C showed the best result.     

Induction of meiotic gynogenesis in bagrid catfish (Pseudobagrus ussuriensis) with homologous sperm and its confirmation for female homogamety

The bagrid catfish, Pseudobagrus ussuriensis, exhibits significant sexual dimorphism in growth rate and body size with males growing faster than females. Therefore, an all‐male culture can dramatically increase the output and profitability of fishery products. According to the monosex breeding route, super‐male individuals’ acquirement by XY male sex reversal and artificial gynogenesis is the key step. An effective protocol to induce meiotic gynogenesis using homologous sperms has been developed in this study. The most effective UV irradiation for sperm genetic inactivation was found to be at a distance of 20 cm with 66 μW/cm² light intensity for 25 min. Optimal treatment for cold shock was 5 min post‐fertilization at 0‐4°C for 30 min, which gave the best survival rate of 13.65 ± 2.87%. The sex ratio in the meiotic gynogens showed a significant female‐biased deviation (p < .05); thirty meiogynogens and their parents were further analysed using a male‐specific AFLP marker, of which only the male parent produced a male‐specific DNA band of 412 bp. These results indicated the female homogametic XX/XY sex determination system in P. ussuriensis. The optimization of a protocol for the successful induction of meiogynogenesis in the bagrid catfish lays the basis for all‐male production and is useful in ascertaining the genetic sex determination system in this promising aquaculture species.     

Induction of heterozygous and homozygous diploid gynogenesis in Betta splendens (Regan) using hydrostatic pressure

Abstract Optimum conditions for hydrostatic pressure treatment for duplication of chromosome set in gynogenetically activated fighting fish, Betta splendens (Regan), eggs were identified. Maximum survival of heterozygous gynogens was 50%, when 2·5-min-old eggs, after insemination with UV irradiated tilapia sperm, were pressure shocked at 7000psi for 6 min. The frequency (21%) of homozygous gynogenetic fry was high, when the 34min (post-insemination) old eggs, which were inseminated with tilapia sperm, were pressure shocked for 5 min. Sex ratio of gynogenetic progeny suggested that the mechanism of sex determination in this fish is homogametic female and heterogametic male.     

Effect of temperature on α-tocopherol, fatty acid profile, and pigments of Diacronema vlkianum (Haptophyceae)

Diacronema vlkianum was grown in polyethylene bags at two different temperatures (18 and 26°C) in the laboratory. The biochemical composition level decreased when the temperature increased from 18 to 26°C. The maximum cell number at 18°C was 11.9 × 10⁶ cells ml⁻¹, while maximum cell number at 26°C was 1.6 × 10⁶ cells ml⁻¹. The maximum level of α-tocopherol was 257.7 ± 21.6 μg g⁻¹ dry weight (DW) at 18°C. The highest total carotenoids and chlorophylls were 6.5 mg g⁻¹ DW and 4.3 mg g⁻¹ DW, respectively, and the main pigments were determined as astaxanthin and lutein. Polyunsaturated fatty acids were found to be the predominant group, reaching 39.5% of the total fatty acids at 18°C. This comprised 20:5(n − 3) as the main polyunsaturated fatty acids (20.4%, at 18°C) followed by 22:6(n − 3) (4.8%, at 18°C). The results suggest that D. vlkianum can be successfully used as feed in shellfish hatcheries or aquaculture hatcheries, either as a substitute or in association with other microalgae, when this algae is cultured at 18°C.     

Greenlip abalone (Haliotis laevigata Donovan, 1808) sperm cryopreservation using a programmable freezing technique and testing the addition of amino acid and vitamin

This study investigated factors key to the development of sperm cryopreservation in the greenlip abalone Haliotis laevigata using a programmable freezing technique, including (1) permeable cryoprotectant agent (CPA) selection; (2) cooling rate; (3) endpoint temperature; (4) thawing temperature; (5) sperm to egg ratio and (6) sugar, vitamin and amino acid supplementation, using sperm motility, fertilization rate, plasma membrane integrity, mitochondrial membrane potential or acrosome integrity as quality assessment indicators. Results showed that among the permeable CPAs evaluated, 10% dimethyl sulfoxide was the most suitable for greenlip abalone sperm cryopreservation. The highest post‐thaw sperm motility was achieved with the sperm being frozen at a cooling rate of ?5°C min^?1 to ?30°C from 0°C and thawed and recovered in 40°C and 18°C seawater baths respectively. The addition of sugars in 10% dimethyl sulfoxide did not significantly improve the post‐thaw sperm motility and fertilization rate. The addition of 0.6% glycine, 0.2% taurine or 0.02% L‐ascorbic acid, on the other hand, significantly improved the post‐thaw sperm motility. However, only the addition of 0.6% glycine improved the post‐thaw sperm fertilization rate, which was further confirmed by the improvement of the post‐thaw sperm mitochondrial membrane potential and acrosome integrity through flow cytometry analysis.