Herpes virus in juvenile Pacific oysters Crassostrea gigas from Tomales Bay, California, coincides with summer mortality episodes

Pacific Crassostrea gigas and eastern C. virginica oysters were examined between June 2002 and April 2003 from 8 locations along the east, west and south USA coasts for oyster herpes virus (OsHV) infections using the A primer set in a previously developed PCR test. Only surviving Pacific oysters from a mortality event in Tomales Bay, California, USA, where annual losses of oysters have occurred each summer since 1993, were infected with a herpes-like virus in 2002. PCR examination using template amounts of both 50 and 500 ng were essential for OsHV detection. Sequence analysis indicated that the Tomales Bay OsHV was similar to that identified in France with the exception of a single base pair substitution in a 917 bp fragment of the viral genome. However, unlike the French OsHV-1, the Tomales Bay OsHV did not amplify with the primer pair of a second OsHV-1 PCR assay, suggesting that further characterization of these viruses is warranted. No evidence of Cowdry type A viral infections characteristic of herpes virus infections or other pathogens were observed in OsHV-infected oysters. Hemocytosis, diapedesis and hemocyte degeneration characterized by nuclear pycnosis and fragmentation were observed in infected oysters, which is consistent with previous observations of OsHV infections in France. Together these data suggest that OsHV may be associated with the annual summer Pacific oyster seed mortality observed in Tomales Bay, but establishment of a causal relationship warrants further investigation.     

Commercial-scale sperm cryopreservation of diploid and tetraploid Pacific oysters, Crassostrea gigas

Cryopreservation of sperm from tetraploid organisms (the possession of four chromosome sets) is essentially unexplored. This is the first cryopreservation study to address sperm from tetraploid Pacific oysters, Crassostrea gigas, and addresses the commercial production of triploid oysters (three chromosome sets). Initial motility, refrigerated storage of undiluted sperm, osmolality of extender solutions, sperm concentrations, equilibration time, and cryoprotectants of propylene glycol and dimethyl sulfoxide were evaluated with sperm from diploid and tetraploid oysters. Unlike most teleost fishes, in which the duration of active motility is typically brief, the motility of sperm from oysters lasts for hours. The present study showed that responses to treatment effects by sperm from tetraploids were different from diploids. The majority of tetraploid experiments resulted in less than 10% motility after thawing and less than 5% fertilization. The highest fertilization obtained for thawed sperm was 96% for sperm from diploid oysters and 28% for sperm from tetraploid oysters. Differential responses to treatments by sperm from tetraploid and diploid oysters may be due to differences in gonadal development. However, the use of cryopreserved sperm from tetraploid Pacific oysters produced 100% triploid offspring by fertilization of eggs from diploid females as determined by flow cytometry of larvae. This study demonstrates that sperm from tetraploid oysters can be collected, frozen, and stored for production of triploid offspring.     

Systematic factor optimization for sperm cryopreservation of tetraploid Pacific oysters, Crassostrea gigas

The availability of tetraploid Pacific oysters provides a unique opportunity for comparative studies of sperm cryopreservation between diploids and tetraploids. In parallel to studies with sperm from diploid oysters, this study reports systematic factor optimization for sperm cryopreservation of tetraploid oysters. Specifically, this study evaluated the effects of cooling rate, single or combined cryoprotectants at various concentrations, equilibration time (exposure to cryoprotectant), and straw size. Similar to sperm from diploids, the optimal cooling rate was 5 degrees C/min to -30 degrees C, followed by cooling at 45 degrees C/min to -80 degrees C before plunging into liquid nitrogen. Screening of single or combined cryoprotectants at various concentrations showed that a combination of the cryoprotectants 6% polyethylene glycol/4% propylene glycol and 6% polyethylene glycol/4% dimethyl sulfoxide yielded consistently high post-thaw motility. A long equilibration (60 min) yielded higher percent fertilization, and confirmed that extended equilibration could be beneficial when low concentrations of cryoprotectant are used. There was no significant difference in post-thaw motility between straw sizes of 0.25 and 0.5 mL. Despite low post-thaw fertilization (<10%) in general for sperm from tetraploids, optimized protocols in the present study effectively retained post-thaw motility for sperm from tetraploid oysters. This study confirmed that sperm from tetraploid Pacific oysters were more negatively affected by cryopreservation than were those of diploids. One possible explanation is that sperm from these two ploidies are different in their plasma membrane properties (e.g., structure, permeability, and elasticity), and the plasma membrane of sperm from tetraploids is more sensitive to cryopreservation effects. The fact that combinations of non-permeating and permeating cryoprotectants improved post-thaw motility in sperm from tetraploids provided presumptive evidence for this interpretation.     

Regeneration of radiation-damaged digestive tissues in juvenile Pacific oysters (Crassostrea gigas)

Juvenile Pacific oysters, Crassostrea gigas, were irradiated with 16 and 40 krad and their tissues examined histologically. Degenerative syndromes and tissue regeneration processes were determined for the stomach, gut, collecting ducts, and digestive tubules. Following degeneration, tissue regeneration was observed in the digestive tissues of most oysters exposed to 16 krad and in a limited number exposed to 40 krad. Regeneration was first observed in the digestive tubules and subsequently in the stomach, gut, and collecting ducts. Cellular repopulation of the digestive tubules involved epithelialization with large, undifferentiated crypt cells which then differentiated into functional secretory and absorptive cells. Regeneration in the stomach, gut, and collecting ducts was initiated by proliferative islands of small basophilic cells. Mitotic division of those cells and their subsequent differentiation into functional epithelial cells resulted in the rapid restoration and apparent recovery of the affected tissues. The results of these studies indicate that radioresistance of juvenile C. gigas may in part be due to the remarkably efficient regenerative mechanisms involved in replacing injured or lost digestive tissues.     

Spatio-temporal variations in biological performances and summer mortality of the Pacific oyster Crassostrea gigas in Normandy (France)

Mortality and biological performances of half-grown Crassostrea gigas were studied from spring 2000 to autumn 2001 at six instrumented stations located in two areas (Gefosse and Grandcamp) of the Bay of Veys (Normandy). Shell and meat growth, condition indexes and a macroscopic maturity index were determined on oysters deployed at the six stations in order to assess spatial variability in the influence of environmental conditions. In addition, histological and biochemical analyses were performed in order to determine the sex and establish the reproductive cycle (at all six sites) and the biochemical composition (at four stations). The data set including monthly mean temperatures and data provided by examination of 2,837 oysters were analysed by Principal Component Analysis and a Hierarchical Ascending Clustering which resulted in the formation of four clusters. The highest station on the shoreline belonged to a cluster characterized notably by low total weight due to a short immersion/feeding period, whereas all other stations belonged to another single cluster. Nevertheless, various biological differences were found between these stations, e.g. the reproductive cycle was generally synchronized throughout the bay but some differences relative to spawning occurrence were observed. In 2000, oyster mortality was higher at Gefosse than at Grandcamp, the latter being a more marine area. In 2001, oyster mortalities were significantly higher and all stations were strongly affected. In the Bay of Veys, oyster biological performances and mortality thus showed spatio-temporal variations which were worthy to be discussed.     

QTL for resistance to summer mortality and OsHV‐1 load in the Pacific oyster (Crassostrea gigas)

Summer mortality is a phenomenon severely affecting the aquaculture production of the Pacific oyster (Crassostrea gigas). Although its causal factors are complex, resistance to mortality has been described as a highly heritable trait, and several pathogens including the virus Ostreid Herpes virus type 1 (OsHV‐1) have been associated with this phenomenon. A QTL analysis for survival of summer mortality and OsHV‐1 load, estimated using real‐time PCR, was performed using five F₂ full‐sib families resulting from a divergent selection experiment for resistance to summer mortality. A consensus linkage map was built using 29 SNPs and 51 microsatellite markers. Five significant QTL were identified and assigned to linkage groups V, VI, VII and IX. Analysis of single full‐sib families revealed differential QTL segregation between families. QTL for the two‐recorded traits presented very similar locations, highlighting the interest of further study of their respective genetic controls. These QTL show substantial genetic variation in resistance to summer mortality, and present new opportunities for selection for resistance to OsHV‐1.     

Cadmium affects the expression of heat shock protein 90 and metallothionein mRNA in the Pacific oyster, Crassostrea gigas

Cadmium (Cd) is a widespread nonessential heavy metal that enters the aquatic environment as a result of natural processes and human activities such as wastewater production, agriculture, and mining. To determine the effects of Cd on organisms, we investigated its time- and dose-related effects on mRNA levels of heat shock protein 90 (HSP90) and metallothionein (MT) in the gill and digestive gland and changes enzyme levels in the hemolymph of the Pacific oyster Crassostrea gigas. Full-length HSP90 cDNA was isolated from C. gigas by rapid amplification of cDNA end (RACE) techniques and found to contain 2154 nucleotides, including an open reading frame, and was predicted to encode a protein of 717 amino acids. BLAST analysis indicated that the HSP90 gene of C. gigas shared high homology with known HSP90 genes of other mollusks. The expression of HSP90 mRNA increased significantly with exposure to 0.01 ppm Cd for 11 days or 0.05 or 0.1 ppm Cd for 7 days. The expression of MT mRNA increased significantly with exposure to 0.01, 0.05, or 0.1 ppm Cd for 11 days. Glutamate oxaloacetate and glutamate pyruvate levels increased significantly with exposure to 0.05 or 0.1 ppm Cd for 7 days. These results indicate that HSP90 and MT play important roles in the physiological changes related to metabolism and cell protection that occur in Pacific oysters exposed to Cd.     

Context-dependent impacts of a non-native ecosystem engineer, the Pacific oyster Crassostrea gigas

The introduction of non-native species represents unprecedented large-scale experiments that allow us to examine ecological systems in ways that would otherwise not be possible. Invasion by novel ecological types into a community can press a system beyond the bounds normally seen and can reveal community interactions, local drivers and limits within systems that are otherwise hidden by coevolution and a long evolutionary history among local players, as well as local adaptation of species. The success of many invaders is attributed to their ability to thrive in a wide range of habitat types and physical conditions, setting the stage for direct examination of ecological impacts of a species across a range of habitat and community contexts. Bivalves are well-known ecosystem engineers, especially oysters, which are the target of wild-caught fisheries and aquaculture. The Pacific oyster, Crassostrea gigas, is grown worldwide for aquaculture, and is presently invading shores on virtually every continent. As a consequence, this non-native species is having large impacts on many systems, but the types of impacts are system specific, and greatly depend on substrate type, how physiologically stressful the environment is for intertidal zone species, and the presence of native engineering species. A novel type of engineering effect is identified for this non-native species, whereby it alters not only the physical environment, but also the thermal environment of the community it invades. The impacts of engineering by this non-native species will depend not only on whether it facilitates or inhibits species but also on the trophic level and ecological role of the species affected, and whether similar ecological types are found within the system.     

In vitro research of anti-HSV-1 activity in different extracts from Pacific oysters Crassostrea gigas

Mortalities related to the detection of Ostreid Herpesvirus 1 (OsHV-1) have been previously reported in France among larvae and spat of the Pacific oyster Crassostrea gigas. Adult oysters appear less sensitive to herpesvirus infections, although OsHV-1 has been detected in adults without signs of disease or mortality. This suggests that the virus is able to persist in its host and that adult oysters may be able to control OsHV-1 infection. Little is known about antiviral substances in invertebrates. The present work concerns the research of antiviral substances in adult oyster C. gigas, where putative antiviral activities were monitored using 3 strategies: (1) in metabolites with variable polarity, (2) in peptidic extracts and (3) in crude haemolymph. In vitro antiviral assays were based on inhibition of Herpes simplex virus type 1 (HSV-1) replication in Vero cell monolayers. All extracts presented no cytotoxicity. Antiviral activity was detected in the fresh filtered haemolymph (EC50:425 microg ml(-1)) and seasonal variation of the haemolymph antiviral activity was monitored.     

Characterisation and genetic polymorphism of metallothionein gene CgMT4 in experimental families of Pacific oyster Crassostrea gigas displaying summer mortality

Summer mortality events have been observed in Pacific oyster Crassostrea gigas for several decades. This paper examines the selective pressure exerted by summer mortality on the polymorphism of a newly identified oyster metallothionein gene. CgMT4 cDNA and genomic sequences were obtained. CgMT4 was studied in two generations of oysters reared in three sites on the French Atlantic coast, using single strand conformation polymorphism analysis. Four alleles were detected. Individuals carrying genotype MT4-CD seem to have higher susceptibility to summer risk conditions. The MT4 gene could be a potential new genetic marker for susceptibility; further validation studies are recommended.     

A Vibrio splendidus strain is associated with summer mortality of juvenile oysters Crassostrea gigas in the Bay of Morlaix (North Brittany, France).

Juvenile oysters Crassostrea gigas cultured in the Bay of Morlaix (France) have suffered unexplained summer mortalities for over a decade. In the present study, we tested the hypothesis that a bacterial pathogen could be responsible for this phenomenon. A first attempt failed to isolate a bacterial pathogen from moribund or weak oysters. Only non-pathogenic, probably opportunistic, bacteria were isolated. As an alternative approach, we focused on oysters presenting reduced stress-response capacities (determined by circulating noradrenaline measurements), a characteristic of juvenile oysters entering an early phase of the disease. Cultures of bacterial isolates on TCBS plates revealed that a Vibrio strain was present in diseased oysters and scarce or absent in healthy oysters. Experimental infections indicated that this Vibrio can cause mortalities of juvenile oysters when injected at concentrations ranging from 10(4) to 10(8) CFU oyster(-1). Similarly to the summer mortality disease, the Vibrio isolate caused higher mortalities at higher temperatures; apparently, it could not be transmitted horizontally, it did not affect adult oysters and it induced stress-response dysfunctions in juvenile oysters. Phenotypic and genotypic characterizations identified the pathogen as Vibrio splendidus. Taken together, the present results satisfy Koch's postulate and suggest that this bacterial strain is probably responsible for the juvenile oyster summer mortalities in the Bay of Morlaix.     

Evidence for colonization and destruction of hinge ligaments in cultured juvenile Pacific oysters (Crassostrea gigas) by cytophaga-like bacteria

Several strains of cytophaga-like gliding bacteria (CLB) were isolated as numerically dominant or codominant components of bacterial populations associated with proteinaceous hinge ligaments of cultured juvenile Pacific oysters, Crassostrea gigas. These bacteria were morphologically similar to long, flexible bacilli occurring within degenerative lesions in oyster hinge ligaments. Among bacteria isolated from hinge ligaments, only CLB strains were capable of sustained growth with hinge ligament matrix as the sole source of organic carbon and nitrogen. In vitro incubation of cuboidal portions of ligament resilium with ligament CLB resulted in bacterial proliferation on the surfaces and penetration deep into ligament matrices. Bacterial proliferation was accompanied by loss of resilium structural and mechanical integrity, including complete liquefaction, at incubation temperatures between 10 and 20 degrees C. The morphological, distributional, and degradative characteristics of CLB isolated from oyster hinge ligaments provide compelling, albeit indirect, evidence that CLB are the agents of a degenerative disease affecting juvenile cultured oysters. The motility, metabolic, and hydrolytic characteristics of hinge ligament CLB and the low moles percent G + C values (32.4 to 32.9) determined for three representative strains indicate that they are marine Cytophaga spp.     

Molecular identification and expression of heat shock cognate 70 (hsc70) and heat shock protein 70 (hsp70) genes in the Pacific oyster Crassostrea gigas

The 70-kDa heat shock protein (Hsp) family is composed of both environmentally inducible (Hsp) and constitutively expressed (Hsc) family members. We sequenced 2 genes encoding an Hsp70 and an Hsc70 in the Pacific oyster Crassostrea gigas. The Cghsc70 gene contained introns, whereas the Cghsp70 gene did not. Moreover, the corresponding amino acid sequences of the 2 genes presented all the characteristic motifs of the Hsp70 family. We also investigated the expression of Hsp70 in tissues of oysters experimentally exposed to metal. A recombinant Hsc72 was used as an antigen to produce a polyclonal antibody to quantify soluble Hsp70 by enzyme-linked immunosorbent assay in protein samples extracted from oysters. Our results showed that metals (copper and cadmium) induced a decrease in cytosolic Hsp70 level in gills and digestive gland of oysters experimentally exposed to metal. These data suggest that metals may inhibit stress protein synthesis.     

Evidence for colonization and destruction of hinge ligaments in cultured juvenile Pacific oysters (Crassostrea gigas) by cytophaga-like bacteria.

Several strains of cytophaga-like gliding bacteria (CLB) were isolated as numerically dominant or codominant components of bacterial populations associated with proteinaceous hinge ligaments of cultured juvenile Pacific oysters, Crassostrea gigas. These bacteria were morphologically similar to long, flexible bacilli occurring within degenerative lesions in oyster hinge ligaments. Among bacteria isolated from hinge ligaments, only CLB strains were capable of sustained growth with hinge ligament matrix as the sole source of organic carbon and nitrogen. In vitro incubation of cuboidal portions of ligament resilium with ligament CLB resulted in bacterial proliferation on the surfaces and penetration deep into ligament matrices. Bacterial proliferation was accompanied by loss of resilium structural and mechanical integrity, including complete liquefaction, at incubation temperatures between 10 and 20 degrees C. The morphological, distributional, and degradative characteristics of CLB isolated from oyster hinge ligaments provide compelling, albeit indirect, evidence that CLB are the agents of a degenerative disease affecting juvenile cultured oysters. The motility, metabolic, and hydrolytic characteristics of hinge ligament CLB and the low moles percent G + C values (32.4 to 32.9) determined for three representative strains indicate that they are marine Cytophaga spp.     

Fixation methods can produce misleading artifacts in sperm cell ultrastructure of diploid and tetraploid Pacific oysters, Crassostrea gigas

Spermatozoa from diploid and tetraploid Pacific oysters (Crassostrea gigas) were examined after anisotonic fixation. Morphological anomalies, such as membrane rupture, detached tails, and the formation of tail vesicles (typically associated with damage attributable to procedures such as cryopreservation) were observed; the Mantel-Haenszel Chi-square test indicated a strong association between the anomalies and fixative osmolality (P<0.001). The present study also indicated that media in a range of 800 to 1,086 mOsm/kg could be assumed to be functionally isotonic to Pacific oysters, and osmolalities below or above this caused severe cell damage. For example, the maximum volume of flagella obtained after hypotonic fixation was approximately twice the volume of the flagella in isotonic fixation. Sperm cell flagellar volumes after hypertonic fixation (1,110 mOsm/kg) were 32% smaller than those in isotonic fixation, and sperm heads were 25% smaller. Although the damage associated with anisotonic fixation was evident in all parts of the sperm cells, the most vulnerable locations were the plasma membrane and flagellum motor apparatus. The formation of tail vesicles after hypotonic fixation was also examined. Because of water uptake, oyster sperm became swollen in hypotonic fixative, and bending or coiling of the axoneme within the tail vesicles led to the appearance of multiple axonemal structures in cross sections when observed by transmission electron microscopy. This phenomenon might be generally misinterpreted as the presence of double tails. This and other fixation artifacts can lead to the misinterpretation of damage caused by cryopreservation in ultrastructure studies of sperm of aquatic species, especially those in marine species.This work was supported in part by funding from the USDA-SBIR program, 4Cs Breeding Technologies, and the Louisiana Sea Grant College Program.     

Reproductive patterns of the Pacific oyster Crassostrea gigas in France

Summary

In France, national management programs focus research on understanding reproductive factors in Crassostrea gigas to confront problems of the oyster industry. However, little information has been documented in which reproductive patterns include sexual changes. The reproductive cycle of oysters at three sites of the Atlantic coast of France was examined from 1996 to 1998, and the seasonal variations in oocyte size-frequencies, and sex ratio were described. The results showed a synchronism within the population concerning reproductive behavior. Young oocytes are generated after spawning and show no apparent changes during winter. Growth of oocytes begins in spring and cells reach maturity in April-May and are ready for a single spawning season in June-July. Oocytes that were not released during spawning are reabsorbed within the gonad. The significant difference between sites is that spawning occurred 1 month later in the southern area. A modal analysis showed that oocyte populations in the sample individuals are primordially bimodal, but with polymodal occurrences in June-July, in some cases. Irregular alternative sexuality was detected at all sites, and hermaphrodites appear to be a transition phase that allows changes from male to female during early spring. Previous observations, together with the study of the development of oocyte cohorts over time, permit a hypothetical model concerning the kinetics of gametogenesis in C. gigas. The model suggests that primary oocytes are generated from energy supplied from degenerating, as well as young oocytes that do not reach the mature stage within the gonad during autumn-winter. It seems that, during vitellogenesis, there is disintegration of smaller cells coupled with transfer of energy to the larger oocytes, which continue to grow and mature.     

Predictive models for the effect of storage temperature on Vibrio parahaemolyticus viability and counts of total viable bacteria in Pacific oysters (Crassostrea gigas)

Vibrio parahaemolyticus is an indigenous bacterium of marine environments. It accumulates in oysters and may reach levels that cause human illness when postharvest temperatures are not properly controlled and oysters are consumed raw or undercooked. Predictive models were produced by injecting Pacific oysters (Crassostrea gigas) with a cocktail of V. parahaemolyticus strains, measuring viability rates at storage temperatures from 3.6 to 30.4°C, and fitting the data to a model to obtain parameter estimates. The models were evaluated with Pacific and Sydney Rock oysters (Saccostrea glomerata) containing natural populations of V. parahaemolyticus. V. parahaemolyticus viability was measured by direct plating samples on thiosulfate-citrate-bile salts-sucrose (TCBS) agar for injected oysters and by most probable number (MPN)-PCR for oysters containing natural populations. In parallel, total viable bacterial counts (TVC) were measured by direct plating on marine agar. Growth/inactivation rates for V. parahaemolyticus were −0.006, −0.004, −0.005, −0.003, 0.030, 0.075, 0.095, and 0.282 log₁₀ CFU/h at 3.6, 6.2, 9.6, 12.6, 18.4, 20.0, 25.7, and 30.4°C, respectively. The growth rates for TVC were 0.015, 0.023, 0.016, 0.048, 0.055, 0.071, 0.133, and 0.135 log₁₀ CFU/h at 3.6, 6.2, 9.3, 14.9, 18.4, 20.0, 25.7, and 30.4°C, respectively. Square root and Arrhenius-type secondary models were generated for V. parahaemolyticus growth and inactivation kinetic data, respectively. A square root model was produced for TVC growth. Evaluation studies showed that predictive growth for V. parahaemolyticus and TVC were “fail safe.” The models can assist oyster companies and regulators in implementing management strategies to minimize V. parahaemolyticus risk and enhancing product quality in supply chains.     

Delayed meiosis and polar body release in eggs of triploid Pacific oysters, Crassostrea gigas, in relation to tetraploid production

The dynamics of polar body release are important for creating polyploid shellfish. For producing triploids, these dynamics concern meiosis in diploid eggs and are well understood. For creating tetraploids, eggs from triploids are employed and the dynamics, variation, and environmental influences upon polar body release are less studied. We investigated the effects of several agents on the timing of 50% first polar body (PB1) release in eggs of triploids. PB1 release is generally slower in triploid eggs than diploid ones at 26 degrees C. Lowering the temperature (from 26 to 19 degrees C) had a marked effect on timing of 50% PB1 in both diploid and triploid eggs. While lower temperature merely slowed development in diploid eggs, it nearly halted it in triploid eggs. At any temperature, the variability in 50% PB1 release was much higher in triploid eggs than diploid ones; this variation occurred both within eggs from individual females and among eggs from different females. The amount of time eggs remain in seawater between the time they are stripped and fertilized (or time of hydration) also affected rate of meiosis. In triploid eggs, the average time necessary for the expulsion of 50% PB1 was 23 min post-fertilization (PF) for 75 min of hydration versus 29 min PF for 35 min. However, increasing the time of hydration had no effect on the variability in the timing among females. Serotonin also had no effect on the dynamics of polar body release in triploids. Variability among triploid females in timing of meiosis cannot be improved with any treatments we tried. Consequently we recommend that treatments of triploid eggs to produce tetraploids incorporate a single female at a time.     

Proteomic identification of quality factors for oocytes in the Pacific oyster Crassostrea gigas

We used a 2-DE proteomic approach to identify abundant proteins linked to oocyte quality in the Pacific oyster Crassostrea gigas, an economically important bivalve. Oocyte quality of 14 females was estimated by recording fertilisation and early developmental success until D-larval stage under controlled conditions. Proteins that were differentially expressed between females showing high or low oocyte quality were identified by nano-liquid chromatography tandem mass spectrometry. Twelve up-accumulated spots associated with low quality oocytes revealed 10 distinct proteins, including vitellogenin - breakdown products and metabolic enzymes. Eight up-accumulated spots from high quality oocytes revealed 6 distinct proteins, including chaperone molecules and cell-cycle control proteins. This is the first proteomic study dedicated to oocytes in C. gigas. Our results improve current knowledge about protein factors associated with oocyte quality in this species, and our understanding of the proteomic processes involved in their developmental competence.