Oyster Eggs Research Articles

Impact of Environment and Ontogeny on Relative Fecundity and Egg Quality of Female Oysters (Crassostrea virginica) from Four Sites in Northern Chesapeake Bay.

Resource allocation to reproduction is a primary physiological concern for individuals, and can vary with age, environment, or a combination of both factors. In this study we quantified the impact of environment and individual age on the reproductive output of female oysters Crassostrea virginica. We determined the relative fecundity, egg total lipid content, and overall and omega-3/omega-6 (ω3/ω6) fatty acid signatures (FAS) of eggs spawned by female oysters over a 2-year period (n = 32 and n = 64). Variation was quantified spatially and ontogenetically by sampling young and old oyster populations from two rivers in Chesapeake Bay, totaling four collection sites. During Year 1, when oysters underwent oogenesis in different locations, overall and ω3/ω6 egg FAS varied significantly by river, with no significant differences observed in the FAS of oysters by age in Year 1. In Year 2, when oysters from different sites underwent oogenesis in a single location, no significant differences in the overall egg FAS or ω3/ω6 egg FAS by river or age were observed. These findings suggest that oysters integrate environment into their reproductive output, but that time spent growing at a specific location (in this case, represented by oyster age) plays a relatively minor role in the biochemical composition of oyster eggs. These results have consequences for our understanding of how resources are allocated from the female oyster to eggs and, more generally, the impact of environment and ontogeny on reproductive physiology.

CONCHAS DE OSTRAS E CASCAS DE OVOS MOÍDAS COMO CORRETIVOS DA ACIDEZ DO SOLO - DOI: 10.13083/1414-3984/reveng.v23n6p584-590

Pesquisas têm sido realizadas com alguns resíduos sólidos que apresentam capacidade de correção da acidez do solo, como materiais alternativos ao calcário, em razão da elevada quantidade em que são descartados no ambiente. Objetivou-se, com a realização deste trabalho, avaliar a potencialidade de cascas de ovos e conchas de ostras moídas como corretivos da acidez do solo e obter a curva de neutralização da acidez de amostras de solo do horizonte B de um Latossolo Vermelho Amarelo (LVA). Avaliaram-se a capacidade dos resíduos como corretivos da acidez de acordo com a legislação vigente. Para a obtenção da curva de neutralização da acidez, os resíduos foram misturados em amostras de 500g de solo, nas doses de 0, 2, 4, 8, 16, 32, 64 e 128 t ha-1 e de 0, 2, 4, 8, 16 e 32 t ha-1 para as conchas de ostras e cascas de ovos, respectivamente. A mistura de solo e resíduo permaneceu incubada por um período de duas semanas, medindo-se, ao final desse período, o pH do solo na sua suspensão com água. Os dados obtidos foram submetidos à análise de regressão, ajustando-se a equação de estimativa do valor de pHem função da dose de resíduos aplicada. Os resíduos avaliados possuem elevado potencial como corretivos da acidez do solo e as doses necessárias para que o pH do horizonte B do LVA alcance o valor de 6,0 são de 5,25 e 5,20 t ha-1, para as conchas de ostra e cascas de ovos, respectivamente.

Effects of Enteromorpha prolifera extract on hatchability of Pacific oyster (Crassostrea gigas) eggs

Enteromorph prolifera was the causative species of green tides in the Yellow Sea from 2007 to 2009.Three different organic solvents which were methanol,acetone and chloroform,were used to extract the dry tissue of E.prolifera,and the effects of extracts on hatchability of Pacific oyster(Crassostrea gigas) eggs were tested.It was found that the methanol and acetone extracts had strong inhibitory effects on hatching of oyster eggs,and their high-concentration extracts could completely inhibit the oyster eggs from developing into D-shaped larvae.The chloroform extract,however,did not show any significant concentration-dependent effects on the hatching rates of oyster eggs.The methanol extract of E.prolifera was further purified by liquid-liquid extraction with ethyl acetate and petroleum ether,and the ethyl acetate phase and petroleum ether phase were also tested for their effects on the hatching rates of oyster eggs.It was found that both ethyl acetate and petroleum ether extract could significantly inhibit hatching of oyster eggs.The results of this experiment suggest that E.prolifera can produce bioactive compounds inhibiting hatching of oyster eggs,and the compounds,probably with high polarity,can be extracted with methanol.However,the structure and characteristics of the bioactive compounds in E.prolifera need to be further studied.

Microbial communities associated with early stages of intensively reared orange-spotted grouper (Epinephelus coioides)

The gut microbiota plays key roles in the health and general welfare of fish larvae, the present study characterized the bacterial communities associated with grouper Epinephelus coioides larvae during a period of 22 days post hatch (DPH) in an intensive hatchery using both cultivation-based and cultivation-independent approaches. Both approaches confirmed that bacteria were present in the gut of larvae before and after the onset of exogenous feeding, and the number of cultiviable bacteria increased gradually from 2 DPH to 22 DPH. A more complex bacterial profile was present in larvae fed fertilizer oyster eggs for 4 days (8 DPH), probably as a result of the onset of exogenous feeding. Interestingly, similar internal microbiota were observed in larvae fed fertilized oyster eggs for 4 days (8 DPH) and rotifers for 2 weeks (22 DPH), although different microbial communities were present in the two feeds. This might suggest that the gut environment of E. coioides larvae selects for a common microbiota, which is more closely related with the rearing water than the two feeds. Therefore, bacterial community of the rearing water may play a critical role in the establishment of gut microbiota of fish larvae and more attention should be paid to its practical modulation by using probiotics. In addition, some potentially beneficial bacteria, such as Lactococcus spp., were the major components of the microbiota associated with fertilized oyster eggs, while these bacteria were not detected in larvae samples.

Development of an Immunological Probe to Quantify Reproductive Effort in the Suminoe Oyster,Crassostrea ariakensis(Gould 1861)

A polyclonal antibody specific to an egg protein of the Suminoe oyster, Crassostrea ariakensis, was developed to assess reproductive effort. After 2 mo of immunization, rabbit antiserum showed strong specificity to the egg protein in an enzyme-linked immunosorbent assay (ELISA). The rabbit antioyster egg immunoglobulin G detected as little as 0.2 µg/mL of Suminoe oyster egg protein by ELISA. The quantity of eggs present in an oyster was estimated using ELISA and was expressed as a gonadosomatic index (GSI). Gonadosomatic index values of Suminoe oysters were assessed monthly from January to July 2007 at the Seomjin River estuary off the south coast of Korea. Histology indicated that most oysters were mature and ready to spawn by the middle of July. Mean GSI values for oysters collected in April, when most female oysters are in early developmental stages, varied between 0.6% and 14.0%. In July, most oysters were ready to spawn and GSI values ranged from 17.5–67.0%, with a mean of 47.7%. The potential fecundity of ripe oysters was determined by dividing the number of eggs, which was estimated by ELISA, by the mean dry weight of a single egg (14 ng); fecundity ranged from 162–910 million eggs. The immunological technique used in this study was affordable and sensitive enough to measure variation in the number of eggs present among gametogenic stages.

Assessment of gamete quality for the eastern oyster ( Crassostrea virginica) by use of fluorescent dyes

Evaluation of sperm motility is the single most widely used parameter to determine semen quality in mammals and aquatic species. While a good indicator for fresh sperm viability, post-thaw motility is not always effective at predicting fertilizing ability. Techniques using fluorescent dyes can assess functionality of mammalian sperm, but have not been widely applied in aquatic organisms. The eastern oyster Crassostrea virginica is an important mollusk in the United States, and cryopreservation protocols have been developed to preserve sperm and larvae to assist research and hatchery production. In this study, protocols were developed to assess sperm cell membrane integrity and mitochondrial function by flow cytometry and to assess viability of eggs by fluorescence microscopy. The fluorescent dyes SYBR 14 and propidium iodide (PI) (to assess membrane integrity) and rhodamine 123 ( R123) (to assess mitochondrial membrane potential) were used to evaluate the quality of thawed oyster sperm previously cryopreserved with different cryoprotectant and thawing treatments. Membrane integrity results were correlated with motility of thawed sperm and mitochondrial membrane potential with fertilizing ability. Fluorescein diacetate (FDA) was used to assess cytotoxicity of cryoprotectant solutions and post-thaw damage to oyster eggs. The results indicated that membrane integrity ( P = 0.004) and thawing treatments ( P = 0.04), and mitochondrial membrane potential ( P = 0.0015) were correlated with motility. Fertilizing ability was correlated with cryoprotectant treatments ( P = 0.0258) and with mitochondrial membrane potential ( P = 0.001). The dye FDA was useful in indicating structural integrity of fresh and thawed eggs. Exposure of eggs, without freezing, to dimethyl sulfoxide yielded higher percentages of stained eggs and fertilization rate than did exposure to propylene glycol ( P = 0.002). Thawed eggs were not stained with FDA (<1%) and larvae were not produced.

Enzyme-linked immunosorbent assay (ELISA) used in quantification of reproductive output in the pacific oyster, Crassostrea gigas, in Korea

Polyclonal antibody developed from egg proteins of the Pacific oysters, Crassostrea gigas, was used in quantification of the eggs. Quantity of the egg protein was measured using ELISA and a weight-normalized gonadosomatic index (GSI) was then established. Histology indicated that the oysters collected from Goseong Bay, Korea, in 2000 initiated gametogenesis as early as late February and spawned mostly in late June to early July when water temperature reached 23–25 °C. A second spawning peak was also observed in late August and the spawning activity continued to late October. Oyster egg proteins could be detected by ELISA in all months except March and November; a few oysters collected during December and January contained a measurable amount of eggs. The highest monthly mean GSI, 0.423, was observed in mid-June when the oysters were ready to spawn. The maximum GSI recorded in this study was 0.667. A positive correlation was found between oyster size and fecundity; the number of eggs increased as oyster biomass increased ( r 2=0.7497). Our data suggest that oysters in Goseong Bay produce 3–196 million eggs during the spawning season. The immunological method developed in this study was sensitive enough to measure a small quantity of eggs and considered to be method of choice for studying bivalve reproduction.

Instantaneous Reproductive Effort in Female American Oysters, Crassostrea virginica, Measured by a New Immunoprecipitation Assay.

An immunoprecipitation assay was developed for measuring instantaneous reproductive effort in female American oysters, Crassostrea virginica. Oysters were injected with 14C-leucine and incubated in situ for 1 to 30 h periodically throughout the annual gametogenic cycle. Gonadal protein labeled with 14C-leucine was precipitated from an oyster homogenate with rabbit anti-oyster egg IgG as the primary antibody. Antibody-oyster egg protein complex was further purified by immunoadsorption with staphylococcal protein A cell suspension. The quantity of oyster eggs was determined by single-ring immunodiffusion. A mathematical model was developed to calculate the instantaneous reproductive rate of oysters and to estimate the number of days required from the initiation of gonadal development to spawning. The oyster population was lightly to moderately infected with a protozoan parasite, Perkinsus marinus. A negative correlation between the intensity of infection and the rate of gonadal production suggests that P. marinus retards the rate of gamete development. The seasonal cycle of gamete production determined by direct measurements of egg protein production was not equivalent to that determined by standard gonadal-somatic index (GSI), except at the most basic level. GSI was highest during the spring spawning peak, but the rate of gamete production was highest in the fall. Accordingly, the two measurements, rate versus standing crop (volume of gonad), reveal a substantially different picture about the details of the spawning season. Estimates of the time required to reach spawning condition ranged from several weeks to 1 or 2 months; these values agree with published estimates derived from less direct methods. Direct rate measurements thus seem to accurately reflect the true rate at which gametic tissue is produced in the field. A positive correlation between oyster size and the estimated days to spawn suggests that larger oysters require longer to prepare to spawn. Furthermore, the range in observed somatic and gametic growth emphasizes the conservatism of somatic growth and the volatility of gonadal growth that is borne out by the results of population dynamics models of oysters.

Morphogenesis of Maternal and Paternal Genomes in Fertilized Oyster Eggs (Crassostrea gigas): Effects of Cytochalasin B at Different Periods During Meiotic Maturation.

Fertilized oyster (Crassostrea gigas) eggs, treated with and without cytochalasin B (CB) at varying periods during meiotic maturation, were examined following 7-aminoactinomycin D (7-AAD) and anti-{beta} tubulin staining for DNA and microtubular patterns, respectively. Electron microscopic observations of untreated fertilized eggs revealed the development of an extensive network of endoplasmic reticulum during germinal vesicle breakdown. Germinal vesicle breakdown was not accompanied by the disappearance of the nucleolus; the latter persisted morphologically unchanged throughout fertilization. The first meiotic spindle [12.4 +/- 1.0 {mu}m (1) x 5.4 +/- 0.55 {mu}m (diameter (d); metaphase plate)] was oriented with its long axis perpendicular to the egg's surface. In contrast, the second meiotic spindle was approximately one half the size of the first [7.4 +/- 0.65 {mu}m (1) x 2.91 +/- 0.29 {mu}m (d)] and initially oriented with its long axis parallel to the egg's surface. Just prior to anaphase II, the spindle rotated so that its long axis became perpendicular to the egg's surface. Following its incorporation into the egg cytoplasm, the sperm nucleus dispersed but did not form a nuclear envelope until the completion of polar body formation. Just prior to pronuclear migration, an array of microtubules assembled around the female pronucleus, and then regressed; concomitantly, a sperm aster formed in conjunction with the centrosome associated with the developing male pronucleus. Following their migration and apposition with one another, both pronuclei underwent prophase as independent structures. Chromosomes from the male and female pronuclei became organized on a mitotic spindle in preparation for first cleavage. In zygotes treated with CB continuously or for just meiosis I, anaphase I occurred; however, a polar body failed to form. Consequently, all the diads were retained within the zygote and became situated on a tripolar spindle. In such cases, anaphase II resulted in the production of a variable number of chromosomal aggregations that developed into pronuclei (3 to 6). In contrast, zygotes treated with CB during meiosis II developed spindles characteristic of untreated specimens; however, polar body formation was blocked, resulting in the development of usually two maternal pronuclei. Morphogenesis of C. gigas fertilized eggs is discussed in reference to similar processes in other species and with respect to strategies of triploid embryo production using CB.

Quantitative measurement of reproductive output in the American oyster, Crassostrea virginica (Gmelin), using an enzyme-linked immunosorbent assay (ELISA)

Abstract. A quantitative gonadal index was developed for oysters, Crassostrea virginica (Gmelin. 1791), using polyclonal antibodies from eggs and sperm. Percoll used in the purification of oyster eggs and sperm greatly improved the purity of antigens compared to filtering the egg or sperm through a fine mesh only. The antigen-antibody reaction was tested with indirect sandwich ELISA using alkaline phosphatase-conjugated goat anti-rabbit igG as a secondary antibody. Rabbit anti-oyster egg IgG and anti-oyster sperm IgG initially exhibited a weak cross-reactivity over somatic tissue. Absoring with acetone-dried oyster tissue powder removed this cross-reactivity. Both antisera exhibited strong specific immunological reactions to oyster eggs or sperm respectively. The quantity of eggs or sperm was measured using ELISA and a quantitative gonadosomatic index (dry wt of egg or sperm/dry wt oyster) (GSI) calculated. GSI from ELISA correlated with gonadal stage measured histologically. Monthly mean GSI of female oysters was highest during late spring to early summer (0·157–0·201) and lowest during early winter to early spring (0·002-0·000). Maximum GSI observed during the study was 0·422 for female oysters and 0·446 for male oysters. Female oysters produce 3·7–65·4 million eggs, with an average of 21·1 million during each spawning. A positive correlation was observed between the number of eggs produced and oyster size; the number of eggs in the gonad increased as oyster size (i.e. total dry wt) increased (r= 0·67); however, the relationship was non-linear. Large oysters contained proportionally fewer eggs. Prevalence of Perkinsus marinus parasitism was high, 90–100%, during the study, as was weighted incidence, 1·33 to 2·67, No statistically significant correlation was observed between infection intensity and the per cent weight of oyster eggs or egg number.

Peptides from sperm acrosomal protein that initiate egg development

How sperm initiate egg development is being investigated with gametes of the marine worm Urechis. Sperm acrosomal protein, previously shown to activate eggs ( Gould et al., 1986, Dev. Biol. 117, 306–318 ; Gould and Stephano, 1987, Science 235, 1654–1656 ), was enzymatically cleaved into soluble peptide fragments. When this mixture was added to eggs they activated, and parthenogenetic cleavage often occurred. An active peptide (P23) was purified from the mixture and its sequence was determined to be Val-Ala-Lys-Lys-Pro-Lys. Synthetic peptide had the same biological activity. P23 induced eggs to undergo the complete sequence of changes that normally follows fertilization, including the fertilization potential, completion of meiosis, and DNA replication. When a sperm centrosome was introduced into eggs by prefertilization without activation, and the eggs were subsequently activated by P23, they developed normally to trochophore larvae (the contribution of another sperm component is not ruled out by this experiment). P23 covalently coupled to bovine serum albumin also activated eggs, showing that it acted on the external surface of the egg. The peptide did not activate sea urchin eggs, but did cause oyster eggs to undergo germinal vesicle breakdown.

Meiosis and fertilization of the Japanese pearl oyster eggs at different temperature observed with a fluorescence microscope.

The events of meiosis and fertilization in Japanese pearl oyster eggs allowed to develop at dif-ferent temperatures (18°C, 23°C, and 28°C) were investigated with fluorescence microscope. Change of matemal and paternal chromatin of eggs at 23°C were as follows. Spawned eggs were at metaphase of first meiosis. Subsequent to sperm penetration most eggs completed first and second meiosis by 15 and 24 min after insemination, respectively. Female and male pronuclei were formed by 30 min after insemination. The chromatin of male and female pronuclei con-densed gradually and were finally observed as two groups of chromosomes. The two groups of chromosomes combined and formed on the metaphas plate for first cleavage. By 62 min most eggs completed first cleavage. It was observed at the higher temperature, that the eggs developed faster. Percentages of eggs which showed abnormal chromosome behavior were higher at 18°C and 28°C than at 23°C. Asynchronous development of eggs was observed in all temperature groups. Asynchrony of eggs development was more apparend at 18°C.

ON THE LARVAL REARING OF THE WHITE‐SPOTTED GREEN GROUPER, Epinephelus amblycephalus (BLEEKER), WITH A DESCRIPTION OF LARVAL DEVELOPMENT

ABSTRACTThe white‐spotted green grouper, Epinenephelus amblycephalus (Bleeker), a popular and cmercially important marine food fish in Hong Kong, has been used for intensive cage culture in coastal water for many years. The major constraints to its large‐scale development are, however, the shortage and uncertain supply of fingerlings from the wild. Research efforts have therefore been directed at producing large supplies of fingerlings, either for culture to marketable size or for restocking.Larvae of E. amblycephalus were reared through metamorphosis in 1000‐liter rectangular tanks covered with a large translucent roof. The newly hatched larvae measured 2.0 mm in total length and possessed a large yolk sac. Effects of temperature and salinity on the yolk sac larvae were examined in laboratory experiments. Recommended temperature and salinity for rearing of the early larvae are 24°C and 25–32 Ppt. In the laboratory larvae were fed fertilized oyster eggs, rotifers, Artemia nauplii, copepods and shrimp meat. The developnent stages from hatching through metamorphosis are described. Three mortality peaks were observed during the experiment. The highest larval survival rate through metamorphosis (i.e., 40 days) after hatching was 25%.

Oyster egg development as related to larval production in a commercial hatchery

The effect of the percentage of egg development on production is analyzed in relation to the corresponding larval growth and setting in the operation of a commercial oyster hatchery. During the production period, the percentage of eggs developing into straight-hinge larvae decreased each month. However, the larvae showed no negative effects in time of growth or setting. Larvae from eggs with only 20% straight-hinge development did as well as larvae from eggs with 100% development. The percentage of egg development to straight hinge is not an indication of the success of the larval population during growth or setting.

Polyspermy inhibition in the oyster, Crassostrea virginica.

Inhibition of polyspermy is a critical response during fertilization which ensures that only one sperm nucleus will fuse with the female pronucleus to restore the diploid state. Oyster (Crassostrea virginica) eggs prevent polyspermy by a process occurring at the cell surface. However, 5 min after fertilization, there are still functional sperm receptor sites available for penetration by supernumerary sperm. Reinsemination experiments indicate that there is no decrease in the number of penetration sites during this interval. Yet, the number of sperm entering eggs is restricted to one per fertilized egg at a sperm:egg ratio of 1000:1. At a sperm:egg ratio of 10(5):1, an average of only six sperm were able to penetrate each egg. Gamete binding experiments indicate that there is a gradual decrease in the number of sperm bound to eggs starting at approximately 75 sec and continuing until all sperm are detached. Since eggs are fertilized within seconds of insemination and this process takes at least 12 min, it is considered an unlikely mechanism for the polyspermy block. There are no ultrastructural correlates to the polyspermy block nor to the unbinding of sperm, such as secretion of cortical granules or fertilization envelope formation. Based on reinsemination experiments, kinetic data, and ultrastructural observations, we suggest a physiological block to polyspermy which prevents fusion of gamete plasma membranes, and is mediated by an inhibitory effect directly upon the sperm.

Hybridization of Epinephelus species

Hybridization was achieved between cultured female white-spotted green grouper ( Epinephelus amblycephalus) and wild male red grouper ( E. akaara). Human chorionic gonadotropin was used to induce maturation, and the eggs were fertilized by the dry method. Larval rearing to juveniles was achieved, feeding fertilized oyster eggs, rotifers, and brine shrimp nauplii.

The uptake of dissolved glycine following fertilization of oyster eggs, Crassostrea gigas (Thunberg)

Embryos of the oyster, Crassostrea gigas (Thunberg), can take up [ 14C]glycine from micromolar concentrations, but unfertilized eggs do not. Activation of uptake mechanisms coincides with the first cell division of the fertilized egg. Uptake of glycine is inhibited by ouabain, an inhibitor of the sodium pump. This study, when combined with earlier work, demonstrates that bivalves utilize dissolved organic nutrients throughout their life span, from fertilized egg to adulthood.

On the induced spawning and larval rearing of milkfish, Chanos chanos (Forskal)

A female milkfish, captured at sea, was injected with two hormonal injections of acetone-dried salmon pituitary powder and human chorionic gonadotropin, plus Vitamin B complex. It was stripped, and produced 128,000 ripe eggs with an average diameter of 1.15 mm. Fertilization rate was 38% following artificial fertilization with milt from an uninjected male. A total of 36,000 larvae hatched (74% of fertile eggs) after 26–32 h at 34 ‰ salinity and 27–32°C. The newly hatched larvae measured 3.4 mm in mean total length and possessed a large yolk sac. The mouth of the larvae opened about 54 h after hatching. The larvae were fed with fertilized oyster eggs, rotifers, copepods, brine shrimp, flour and prepared feed, together with Chlorella. A critical period was between the 4th and 6th days with mortality over 80%. The larvae started increasing in length by Day 8, and had the appearance of the wild fry by Day 11. On Day 13 a pigmentation pattern developed and the biggest larva measured 10.0 mm. By Day 18 the larvae measured 12.5 mm, and 14.5 mm by Day 21. A total of 2,859 fry was obtained; the highest larval survival rate obtained from different experimental groups was 46.8%.

Ultraviolet light disinfection of shellfish hatchery sea water: I. Elimination of five pathogenic bacteria

Filtered sea water was seeded with one of five oyster-pathogenic bacteria to obtain a final concentration of 10 5 cells/ml. UV irradiation at a dose of 93 312–155 520 μW s −1 cm −2 rendered the contaminated sea water safe for rearing fertilized eggs of Crassostrea virginica. Fertilized oyster eggs were reared until the larvae began to set in filtered, UV-irradiated sea water and in filtered, non-UV-treated sea water. The data suggest that when a pathogenic bacterium is present the potential for disease is much greater in non-UV-treated sea water.

CONVENTIONAL AND EXPERIMENTAL APPROACHES TO HYBRIDIZATION AND INBREEDING RESEARCH IN THE OYSTER

AbstractExperimental manipulation of oyster eggs and cleavages is being used to support conventional research on inbreeding and hybridization. Twelve crosses of foundation stock in the commercial American oyster (Crassostrea virginica) were made to establish traditional full‐sib lines. In an experimental approach to producing inbreds some parthenogenesis was observed in eggs stimulated with spawned oyster sperm that had been X‐irradiated at doses ranging from 10,000 R to 225,000 R. Geographic and interspecific hybrid crosses with C. virginica have been made. The performance of hybrid larvae stressed with abnormally high and low temperatures was tested. Generally, there was better survival and growth of C. virginica geographic hybrids than of interspecific hybrids with C. gigas. Overall, however, there was better survival and growth of local controls in both experimental hybrid crosses and those crosses made specifically for testing the response of larvae to stress. Cytogenetic data suggested normal fertilization and meiosis in these hybrid crosses. The feasibility of controlling mass fertilization by temporarily inhibiting fertilization in spawning groups intended for hybridization was investigated using EDTA.