Quantification of eggs and sperm in the Black-lip pearl oyster Pinctada margaritifera using an enzyme-linked immunosorbent assay (ELISA)

We have developed immunological probes to quantify eggs and sperm of the Black-lip pearl (BLP) oyster Pinctada margaritifera. The western blot assay revealed that the polyclonal antibodies developed in this study specifically recognized only egg and sperm proteins. These polyclonal antibodies also showed high sensitivities to the antigens, detecting 0.31–10 μg/ml of the egg or sperm proteins in an indirect enzyme-linked immunosorbent assay (ELISA). Accordingly, we used an indirect ELISA to quantify the eggs and sperm in BLP oysters collected in December 2009 from Weno Island in Micronesia and in May 2010 from Tahiti. The gonad somatic index (GSI), a ratio of gonad weight to somatic tissue weight, of the females collected from Weno Island ranged from 3.6 to 18.4 %, while the GSI of the females collected from Tahiti ranged from 5.6 to 12.6 %. Similarly, the GSI of the male BLP oysters from Weno Island ranged from 0.8 to 8.5 %, while the GSI of the males from Tahiti ranged from 4.8 to 7.5 %. These results lead to the conclusion that the immunological probes developed in this study can be successfully applied to quantify BLP oyster gonadal tissues and that these probes can be used in studies of BLP oyster reproductive ecology based on their sensitivity, rapidity, and affordability.     

Gametogenetic cycle and reproductive effort assessed by two methods in 3 age classes of Pacific oysters, Crassostrea gigas, reared in Normandy

Two methods were used to estimate the reproductive output of female Pacific oysters reared in Normandy: histology with image analysis and ELISA (Enzyme-Linked ImmunoSorbent Assay) which allowed the quantification of egg protein. Condition indices, gonad area and gametogenetic stages of the oysters were determined in the entire population (males and females) between May and October 2005. All investigations were performed in 3 age classes: oysters in their first, second or third years (corresponding to spat, half-grown and market-sized oysters, respectively). Both quantitative histology and ELISA provided similar results in terms of reproductive effort (illustrated by the gonado-somatic index, GSI) except during the GSI drop, corresponding to spawning, which was less marked with the ELISA method. Growth depended on oyster age, the sex ratio was well balanced and the reproductive cycle was synchronized in all age classes. In the 3 age classes, most of the oysters were ripe and ready to spawn on August 8, and ten days after the post-spawning stage was observed in 40% of spat oysters and 70% of half-grown and market-sized bivalves. The major difference between age classes was observed in the reproductive investment, with spat having a lower reproductive output. For example, in males and females, the gonad area reached 78–79% in the median animal section at full maturity (August 8) in half-grown and marketable oysters while it attained only 59% in spat. At the same time, GSI in females was, respectively in spat and the 2 oldest age classes, 33% (quantitative histology)–36% (ELISA) and 55% (quantitative histology)–60% (ELISA). The mean assessed gonad weight and fecundities increased with the age of the oysters: 1.3 g and 12 million eggs, 7.8 g and 135 million eggs, and 11.5 g and 146 million eggs in spat, half-grown and market-sized oysters, respectively. Marked differences thus appear between 2 and 3-year-old oysters and spat. As early as their first reproductive cycle, the young oysters not only showed the reproductive features of the species in Normandy, but also a pronounced lower reproductive effort. This lower energy demand could explain their higher survival rate.     

Application of sperm cryopreservation in selective breeding of the Pacific oyster,Crassostrea gigas (Thunberg)

The robustness of Pacific oyster, Crassostrea gigas (Thunberg), sperm cryopreservation in the context of selective breeding based on family lines was investigated. Irrespective of egg density, high fertilization success was achieved with cryopreserved sperm when sperm:egg ratios of 1000:1 to 10 000:1 were used. Variation among replicate runs on the same oyster batches was minimal, indicating that cryopreservation and larval rearing procedures were repeatable. Twenty independent single male–female crosses were made to assess the utility of cryopreserved sperm in selective breeding. The fertility of unfrozen sperm was generally a poor predictor of cryopreserved sperm fertility. Based on D‐larval yields, 17 of the 20 crosses were likely to yield adequate spat for selective breeding (>10⁵ D‐larvae from 1 million eggs), two were marginal (5 × 10⁴ D‐larvae) and one was inadequate (4 × 10³ D‐larvae). An alternative fertilization strategy to improve D‐yield from a given number of sperm was then tested. Fertilizing 10 million eggs at a sperm:egg ratio of 200:1 increased the total D‐yield when compared with fertilizing 1 million eggs at a sperm:egg ratio of 2000:1 for the same male–female pair. We conclude that, despite wide variation in fertility, cryopreserved sperm is useful for family production.     

Influence of gonadal development on metal accumulation in the Portuguese oyster,Crassostrea angulata,in subtropical areas

Metal enrichment makes oyster considered to be an appropriate food source for trace elements, but excessive accumulation can threaten human health. Reproduction is an important biotic factor that affects the metal accumulation in oysters. However, the influence of continuous gonad development model on metal accumulation in some subtropical oysters is still unclear. The present study addresses the concentrations of trace metals and glycogen associated with the gonadal and somatic tissues throughout the reproductive cycle of diploid and triploid Crassostrea angulata cultured in Fujian, China. Cu, Zn and Pb contents of gonads were negatively correlated (p < 0.05) with gonadosomatic index (GSI). The correlation between Cd content and GSI was not significant. There was a positive correlation (p < 0.05) between GSI and Mn content in female gonads while the correlation was negative (p < 0.05) in males, indicating Mn plays an important role in gametogenesis of females. The effects of gonadal maturity and gender on metal accumulation in somatic tissue were not significant (p > 0.05). The concentrations of all metals in sterile triploids were not significantly different from those of the somatic tissue of diploids. Gonadal glycogen content showed a negative correlation with the GSI. However, somatic glycogen content was not affected by gonadal development, indicating that the energy consumed during gametogenesis comes mainly from recently ingested food rather than from energy stored in somatic tissue. The effects of the gonadal development on the metal accumulation in oysters varied according to the metal and tissue type.     

Effects of temperature and photoperiod on the conditioning of the flat oyster (Ostrea edulis L.) in autumn

The production of the flat oyster Ostrea edulis (L.) natural spat in Europe has decreased almost by 60% in the past ten years. Thus, the importance of the production of oyster spat in hatcheries is evident. One of the critical steps in hatchery production is broodstock conditioning, especially difficult in autumn, when gonadal development is in resting period. Conditioning is influence by temperature, photoperiod and nutrition. In this work, the effects of two temperature and three photoperiod regimes on the conditioning of O. edulis were studied for three years by stereological analyses and registering number and dates of spawning and larval yield. Temperature had a positive effect on the gonadal development of O. edulis during conditioning. The percentages of germinal cells in oysters conditioned with a gradient of temperature (14–18°C) were double compared to oysters conditioned at 15°C. Oysters conditioned with longer photoperiods showed higher percentages of germinal cells. There was no interaction between temperature and photoperiod. Spawning was observed in the oysters treated with daylight (8–16 h) ten weeks from the beginning of conditioning. Flat oysters conditioned with 8 h and 8–12 h of daylight delayed the first spawning for a month. Total larval production was higher in the oysters treated with the longest daylight gradient. Gonadal and gametogenic development was a non‐synchronic process and the spawning extended for around two months. A protocol for flat oyster broodstock conditioning in autumn by using both a gradient of temperature (14–18°C) and daylight (8–16 h) is proposed.     

Tetraploid induction in tropical oysters,Crassostrea belcheri (Sowerby) and Crassostrea iredalei (Faustino)

Tetraploid induction has been conducted on temperate oysters but not on tropical oysters. In this study, different heat shocks (32, 35 and 38°C) and cold shocks (1, 4 and 7°C) were used to induce tetraploidy in two tropical oyster species, Crassostrea belcheri and Crassostrea iredalei, through meiosis I inhibition. Temperature shocks were applied on the newly fertilized eggs at 8–10 min post fertilization and terminated when second polar bodies began to form in the control eggs. The ploidy of the larvae and spat was determined via direct chromosome count. The percentage of larval survival until Day 20 was low (between 0.4% and 42.9%) for both temperature shocks and oyster species. No surviving larva was recorded for induction at 1, 4 and 38°C. Tetraploid spat was only recorded in C. iredalei but the percentage is low through heat shock induction of 32 and 35°C. This study shows that the tetraploid induction success rate was slightly higher in C. iredalei compared to C. belcheri. No surviving tetraploid spat were recorded for both oyster species through the cold shock method. This study shows that heat shock can be used to inhibit meiosis for the production of tetraploids but more experiments need to be conducted to determine the optimum temperature when dealing with tropical oysters.     

Cryopreservation of sea bass (Dicentrarchus labrax) spermatozoa in experimental and production simulating conditions

A sperm cryopreservation protocol adapted from turbot, was tested on sea bass using either 250-μL straws or 1.5-mL cryovials. A dilution to 1/3 in Mounib s extender and a cooling rate of −65 °C·min⁻¹ allowed frozen sperm to recover an initial motility similar to that of fresh sperm at thawing; however, significant differences in motility (P < 0.001, n = 10 fish semen) were observed at further post-activation times, the motility decrease being faster in thawed sperm. At the experimental scale, triplicate inseminations of 2-mL aliquots (approximately 2 000 eggs) showed a significant fertility decay of thawed sperm compared to that of fresh sperm (P < 0.01, n = 12 fish semen) when a discriminating 35·10³ spermatozoa to egg ratio was applied. When 70·10³ and 200·10³ spermatozoa per egg were provided in the same experimental conditions, no significant difference appeared between the fertilisation rates of fresh and thawed sperm. In order to validate the procedure for production or cryobank purpose, a scaled-up protocol was established. Two and 50 mL batches of eggs (approximately 2·10³ and 50·10³ eggs, respectively) were inseminated in triplicate using either fresh or thawed individual sperms of 5 males with 200·10³ spermatozoa per egg. The mean fertility decreased by 23.5 % due to cryopreservation. This decline was explained by the loss of fertility of only one sperm, and only in large-volume conditions, probably due to the delay of use after thawing.     

Artificial fertilization in turbot, Scophthalmus maximus (L.): different methods and determination of the optimal sperm–egg ratio

With the aim of improving artificial fertilization (AF) in turbot, Scophthalmus maximus (L.), a series of fertilization experiments was carried out under dry conditions and different wet conditions (eggs/sea water: 2V/V and V/V). Another series of fertilization experiments was carried out with different quantities of sperm pool to determine the optimal ratio of spermatozoa to eggs for each AF method. Sperm pool from two males and eggs from spawns with a viability rate of > 70% were used. The sperm pool’s density (0.4–5.18 × 10⁹ sperm mL^–1) and motility (1–5) had been assessed previously. Significantly different fertilization rates were found when comparing 2V/V and V/V wet conditions. Significantly higher fertilization rates were found in dry fertilization when the sperm–egg ratio was > 9000 spermatozoa per egg and, under wet condition V/V, at 3000–4000 spermatozoa per egg.     

Biochemical composition and quality of captive-spawned cobia Rachycentron canadum eggs

Interest in the commercial production of cobia Rachycentron canadum continues to rise as additional insight is gained into the hardy and fast growing nature of this species. However, research regarding the biochemical composition of captive-spawned eggs and egg and larval quality remains scarce. Such data is essential as a common bottleneck to production is a steady supply of fingerlings for grow-out. This study quantified the biochemical composition and quality of cobia eggs produced over 2 spawning seasons by broodstock on a traditional ‘trash fish’ diet which is commonly fed to tank spawning cobia. Throughout the study, batch fecundity, proportion of floating eggs and percent hatch averaged > 1 million eggs,  0.8 and 70%, respectively. Batch fecundity was significantly higher during the second spawning season as a result of the increased size of the females which weighed 18/22 kg and 22/26 kg at the beginning of each season. A positive correlation was found between the proportion of floating eggs and hatch rate for both spawning seasons. No correlations were found between egg composition (total lipid (30.0 ± 1.1% dry wt), protein (25.4 ± 2.2% dry wt), carbohydrate (2.4 ± 0.3% dry wt), vitamin E (10.2 ± 0.6 μg/g wet wt) or dry weight (119.1 ± 5.5 μg/egg)) and egg quality (proportion of floating eggs, hatch rate, larval growth and larval survival). Further, no differences in egg composition were noted between seasons or over the course of each season. The fatty acid composition of cobia eggs varied between seasons possibly due to changes in the quality of frozen feed (fish, shrimp, squid) given to the broodstock. The only correlation between the fatty acid profile and egg quality was a decrease in the proportion of floating eggs as the total amount of n-3 highly unsaturated fatty acids increased. No relationship between egg quality and amino acid content was noted with the most prominent amino acids being glutamate, leucine, alanine, proline, lysine and aspartate nor were any differences detected between spawning seasons.     

Lipid class and fatty acid composition of eggs of Atlantic halibut, Hippoglossus hippoglossus (L.), in relation to egg quality in captive broodstock

Abstract. Lipid class and fatty acid analyses were carried out on eight batches of pre-fertilized eggs of Atlantic halibut, Hippoglossus hippoglossus (L.). These batches were classified as ‘viable’ or ‘non-viable’ according to fertilization rate, blastomere symmetry and incubation success. The lipid class and fatty acid compositions were very similar between egg categories with exception of cholesterol which was shown to be significantly greater in the ‘non-viable’ eggs. Polar lipid classes predominated (67·1% and 67·0% of the total lipid for ‘viable’ and ‘non-viable’ egg batches respectively), with phosphatidylcholine (PC) being the major class (43·2% and 43·5%) and triacylglycerol (TAG) the main neutral lipid class (18·8% and 17·8%). Gas liquid chromatography of fatty acid methyl esters from the total lipid revealed no significant differences in fatty acid composition. The fatty acid profiles of viable egg batches showed five major fatty acids 16:0 (17·3%), 18:0 (4·8%), 18:1n-9 (9%), 20:5n-3 (119%) and 22:6n-3 (25·9%). The results are discussed in terms of the potential role of lipids as egg quality determinants in fish.     

Lipids in eggs from first-time and repeat spawning Atlantic halibut, Hippoglossus hippoglossus (L.)

Abstract The production of viable larvae is critical to any aquaculture venture. Since lipids are important components of fish eggs, the amount and nature of the lipid may be crucial to the survival of the larvae. In this study the lipid content of unfertilized eggs from first-time and repeat spawning Atlantic halibut females was compared. The egg lipid content for the first-time spawner ranged from 0·18 to 0·41mg/egg; the range for the repeat spawner was 0·27 to 0·38mg/egg. However, the mean total lipid per egg values were similar for both fish at ca. 0·31mg/egg. The repeat spawner prodticed more eggs than the first-time spawner, thus allocating more lipid to egg production. In the eggs from the two fish 62–84% of the lipids was phospholipid.     

Small-scale fry production systems for Nile tilapia, Oreochromis niloticus (L.)

Abstract Experiments were conducted to evaluate and compare the production, quality and survival of eggs and the subsequent growth and survival of fry in two small-scale production systems: natural incubation and egg collection followed by artificial incubation. Artificial incubation produced a significantly greater number and biomass of fry per unit weight of female broodstock (P < 0·05). This was due to the significantly decreased spawning interval in females from which eggs were collected (mean = 23· days) compared to naturally incubating females (mean = 36·8 days). It is also hypothesized that survival during artificial incubation (85%) is greater than in natural incubation. Cost-benefit analysis demonstrated a threefold increase in total net returns for artificial incubation.     

The effects of dietary supplements of polyunsaturated fatty acid on pearl oyster,Pinctada margaritifera L., gonad composition and reproductive output

Black‐lip pearl oyster, Pinctada margaritifera broodstock was collected from the wild. Egg production, hatching rate and larval development were compared between oysters induced to spawn within 2 days after collection in the wild (T1), oysters fed a pure microalgae diet during 24 days before spawning (T2) and oysters fed the same microalgal diet in which 10% of the algae were replaced with 2 μm polyunsaturated fatty acid (PUFA)‐rich microspheres (T3). Administration of lipid microspheres resulted in larger sized eggs, a higher percentage of D‐larvae and larger sized 48‐h‐old larvae (P<0.05). The total and neutral lipid contents of the gonad increased after oysters were fed with microalgae only or with supplementary diet. The major neutral and polar fractions of saturated fatty acid (SFA) were 16C and 18C fatty acids, and not influenced by the diet (P>0.05). The gonads of oysters fed supplementary PUFA contained more docosahexaenoic acid (DHA) and less monounsaturated fatty acids. Higher level of DHA in gonads of T3 was associated with oogenesis and embryogenesis success. The n‐3/n‐6 ratio in the neutral lipid fraction provides a good indication of the spawning condition and predicting egg size and hatching rate.     

Changes in fatty acid and sterol composition during oogenesis in the pearl oyster Pinctada margaritifera

The fatty acid and sterol composition of the oyster Pinctada margaritifera during oogenesis and in eggs was analysed. No major differences were observed during oogenesis, but the egg composition was significantly different from that of gonads. The amount of saturated fatty acids was the highest in eggs and the C16:0 predominant (P<5%); by contrast, the amount of 22:6(n‐3) was significantly lower (P<5%) than in gonads. No major differences were observed for the polar lipid (PL) composition during oogenesis. The main free sterols in gonads and eggs were cholesterol and brassicasterol. Among free sterols, the proportion of cholesterol diminished continuously from the beginning to the end of gonad maturation, and this decrease persisted in eggs after spawning. Cholesterol represented 40% to 55% of the sterol ester encountered in gonad and eggs. This study allowed us to investigate the fatty acid and sterol composition during oogenesis of the pearl oyster P. margaritifera, leading to a clearer understanding of the nutritional requirements of pearl oyster during the reproduction process.     

Farming triploid oysters

Although the commercial benefits of triploidy have been evaluated in the Pacific oyster Crassostrea gigas (Thunberg, 1793), eastern oyster C. virginica (Gmelin, 1791), Sydney rock oyster Saccostrea glomerata (Gould, 1850) and European flat oyster Ostrea edulis (Linnaeus, 1750), so far this technique has only been commercialised for Pacific oysters.

Commercial production of triploids on the West Coast of North America began in 1985. Since then production of triploids has greatly increased and the use of tetraploid males to fertilise eggs from diploids to produce batches of 100% triploids has been developed. In 1999/2000, triploid Pacific oysters made up 30% of all Pacific oysters farmed on the West Coast of North America. Some hatcheries now use tetraploid males instead of chemical or physical stress to produce triploids. The rapid uptake of triploid and tetraploidy techniques has been facilitated by the almost total dependence that these oyster industries have on hatcheries for the supply of seed. This industry in the Pacific Northwest of the US and in British Columbia, Canada, would not have developed to its current size without hatchery seed supplies. Triploids are preferred over diploids in summer because diploids are less marketable when in spawning condition.

There was only limited interest in triploidy production in France until 1999, when IFREMER began to make sperm from tetraploids available to commercial hatcheries. In 1999/2000, only 10% to 20% of all the hatchery-supplied Pacific oyster spat were triploids, but with the use of sperm from tetraploid oysters, this could increase sharply.

Elsewhere around the world, the commercial uptake of triploid oysters has been slow to develop. However, in countries where the production of Pacific oysters is based on hatchery supply of seed, it is likely that with the use of tetraploid oysters, the farming of triploid oysters will increase in the near future.     

Parasites, pathological conditions and mortality in QX-resistant and wild-caught Sydney rock oysters, Saccostrea glomerata

Differences in the survival of QX-resistant fifth generation (QXR₅) and wild-caught (Wild-Caught) Sydney rock oysters were assessed over the QX-disease risk period in the Pimpama River, SE Queensland. Cumulative mortality of Wild-Caught oysters (31.7%) was significantly greater than QXR₅ oysters (0.0%) over the 118 days of the experiment. PCR and histological results showed that Wild-Caught oyster did not die from QX disease. Histology revealed oysters were infected with disseminating hemocytic neoplasia, a Steinhausia-like infection, a Rickettsia-like organism infecting epithelial cells of the gill, digenean flukes encysted in the gonadal tissue and a gill response to an unknown toxin. The cause of mortality is attributed to disseminating hemocytic neoplasia.     

Energy content and chemical composition of gilthead seabream, Sparus aurata L., eggs

Abstract. Gross energy, ash content and elemental composition of carbon, nitrogen and hydrogen in eggs of the gilthead seabream, Sparus aurata L., have been determined throughout several spawning seasons. Also, changes in the biochemical components during embryonic development have been analysed. Gross energy of seabream eggs was approximately 1 J per egg. Dry weight of individual eggs at fertilization varied from 39 to 50μg, the chorion accounting for about 20% of total dry weight. However, there were no differences in carbon, nitrogen, hydrogen and ash percentages. From fertilization to hatching eggs lost 7·2% of total energy (chorion not included). The possible relationship between egg energy content and recent emerged larvae is discussed.     

Production of inbred larvae through self‐fertilization using oocytes and cryopreserved sperm from the same individuals after sex reversal in eastern oyster Crassostrea virginica

The eastern oyster Crassostrea virginica can change sex which makes self‐fertilization possible if sperm can be cryopreserved. In this study, small (~1 year old) and large (~2–3 years old) oysters were biopsied for sperm collection. Survival of the biopsied oysters after 1 year was 50% for small oysters and 17% for large oysters. Oocytes were collected from sex‐reversed females, and self‐fertilized with cryopreserved sperm. Of the 24 cryopreserved samples, 14 individuals had ≤1% fertility when crossed with oocytes from unrelated females, indicating that the cryopreserved sperm had reduced fertility. The other 10 individuals had a fertility of 39 ± 25% when crossed with oocytes from unrelated females (non‐selfing), but showed a significantly lower success of self‐fertilization (12 ± 16%) (P = 0.008), while aliquots of the same oocytes had a fertilization of 83 ± 11% when crossing with fresh sperm. Larvae were produced at day 3 in the self‐fertilized families (12–94% of the fertilized oocytes), and survived to eyed‐larvae stage at days 11–14. Genotyping with 9 microsatellite markers confirmed that the larvae resulted from self‐fertilization in four families. This study demonstrated the feasibility of creating self‐fertilized inbred lines of oysters by use of non‐lethal sperm collection and cryopreservation.     

Impacts of Lactobacillus plantarum in Depuration for Reducing Vibrio parahaemolyticus in Pacific Oysters (Crassostrea gigas)

This study investigated potential application of lactic acid bacteria (LAB) in depuration for reducing Vibrio parahaemolyticus in oysters. Lactobacillus plantarum ATCC 8014, which exhibited strong bactericidal effects against V. parahaemolyticus in vitro, was added to artificial seawater for depuration of Pacific oysters (Crassostrea gigas) inoculated with V. parahaemolyticus BE 98-2029 (O3:K6) to levels of about 10⁴ MPN/g at 15 ± 1 and 10 ± 1°C. Application of L. plantarum ATCC 8014 treatment (10⁷ CFU/mL) in oyster depuration did not enhance reductions of V. parahaemolyticus in oysters depurated at 15 ± 1°C but significantly decreased (p < 0.05) levels of V. parahaemolyticus in oysters depurated at 10 ± 1°C after 5 days (3.40 log reductions) when compared with controls (2.75 log reductions). It is not clear if a competitive exclusion by LABs to compete with V. parahaemolyticus binding sites in oyster tissues plays a role in the reduction of V. parahaemolyticus in the oysters. Further studies utilizing different types of LABs in oyster depuration might provide additional knowledge for application of LAB in depuration for decontaminating V. parahaemolyticus in oysters.     

Optimal egg viability storage conditions in two commercial fairy shrimps (Crustacea: Branchiopoda: Anostraca) from Thailand

Optimal egg viability storage conditions for two commercial fairy shrimps, Branchinella thailandensis and Streptocephalus sirindhornae, were investigated. Eggs for each treatment were kept under eight different conditions at four different temperatures for 24 months. Every 2 months, representative eggs were hatched to evaluate hatching percentages. Low temperature and oxygen, darkness and dry conditions significantly influenced egg hatchability of both species. Undehydrated eggs hatchability in all conditions ranged from 0 to 10% in S. sirindhornae and 0 to 20% in B. thailandensis. Hatching percentages of B. thailandensis were higher than those of S. sirindhornae. Hatchability under all experimental regimes continuously decreased over time, except for dry eggs stored at ?18°C in dark‐anoxic conditions. Hatchability was markedly delayed in both species over time. We demonstrate that both species’ eggs can be stored at ?18°C at least for 24 months with high hatchability in B. thailandensis (80%) and S. sirindhornae (60%) under dry, dark, anoxic conditions.