Digital Image Analysis of Lipid and Protein Histochemical Markers for Measuring Oocyte Development and Quality in Pearl Oyster Pinctada Mazatlanica (Hanley, 1856) (original) (raw)
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Journal of Shellfish Research, 2009
We evaluated indicators of quality for female gonads (ovary and oocytes), male gonads (testis and seminal tubules), and selected somatic tissues (fiber packages in the adductor muscle and adenomeres in the digestive gland) that participate in reproduction of the pearl oyster Pinctada mazatlanica. The goal was to identify timing of optimal broodstock condition for larval rearing practices. Tissue samples were collected seasonally and processed with a combination of histochemistry and digital image analysis to develop a gonad tissue index, a lipid index, and a glycogen index. Seasonal changes in these indicators were correlated with changes in water temperature and chlorophyll a in the water. P. mazatlanica uses a combination of stored reserves and food supply (conservative vs. opportunistic strategy) to regulate reproduction, but the way energy is acquired and allocated varies between sexes. Female gonads contained higher lipid contents during spring. We suggest energy allocation from digestive gland, because this tissue showed lower lipid contents in the same season (conservative strategy). Within oocytes, the accumulation of lipids occurred from nutrients obtained from food supply during winter (19.6°C and ;650 ng/L) (opportunistic strategy). Male gonads contained higher glycogen contents in spring. A decreasing trend in the glycogen content of the adductor muscle was also detected in spring. This suggests that sperm build-up occurs partly at the expense of the glycogen stored in this tissue (conservative strategy). In seminal tubules, no correlation with the glycogen content of adductor muscle was detected, suggesting that these reserves were obtained from food supply in spring (22°C and ;400 ng/L) (opportunistic strategy). Optimal broodstock condition occurs mainly in spring and secondly in early winter.
Tools of histochemistry and digital image analysis were used to quantify changes in the coverage area of lipid droplets (lipid content) of oocytes of the penshell Atrina maura during oogenesis and to determine its relation to changes in water temperature and seston content. These data led to calculating a lipid index as a criterion of gamete development and quality. Gonads were collected monthly for 18 mo and prepared for histochemical processing with Sudan Black B for identification of lipids. Finished slides were digitized for determining stages of oogenesis and variations in the size of oocytes. Two periods of greatest reproductive activity occurred during the study, with a lower peak from November through January (;15°C; 26 mg/g) and a major peak from April through June (;20°C; 25-40 mg/g). Oocyte area significantly varied during the stages of active development (516-2,743 mm 2 ), ripeness (1,073-2,930 mm 2 ), spawning (145-2,939 mm 2 ), and atresia (331-2,001 mm 2 ). Lipid incorporation into oocyte cytoplasm followed a clear seasonal pattern, peaking again in winter and spring. Temporal variations in the lipid index and its relation to oocyte diameter were irregular, but also peaked in winter and spring. Histochemistry and digital image analysis resulted in reliable methods for estimating oocyte development and quality in this species, and can certainly be applied in studies of reproduction of other bivalve, invertebrate, and vertebrate species.
Seasonal biochemical variations in Pacific oyster gonadal tissue during sexual maturation
Fisheries Science, 2000
The morphological profile and changes in biochemical composition of the ovary and testis of the Pacific oyster Crassostrea gigas cultured in Onagawa Bay during maturation were examined. As sexual maturity progressed, the glycogen content in the ovaries and testes decreased. The increases in the protein content in the ovaries corresponded with increases in oocyte diameters. The largest variations in gonadal lipids during sexual maturation occurred with the triglycerides. An increase in the ovary was associated with oogenesis and a decrease in the testis was associated with spermatogenesis. The RNA content of the ovaries was consistently greater than that of the testes, while the DNA content was lower. The RNA content and the RNA/DNA ratio are good indicators of sexual maturity in the ovary; the increasing RNA/DNA ratio in the ovary appears to show the rising synthetic activity of vitellin as one of the proteins produced within the ovary.
Ensuring supplies of pearl oyster spat for commercial grafting operations in Mexico is an ongoing problem. This has refocused research toward improving hatchery propagation protocols. Since gender plays an important role in the physiology of bivalves, we studied the use of fatty acids in the gonad and digestive gland of male and female winged pearl oyster (Pteria sterna) over its natural breeding season. Sampling included two peaks of ripening (February and April 2009), a pre-reproductive period (November 2008), and a post-reproductive period (June 2009). We found a significant increase in storage of docosapolyenoic fatty acids during development and ripe stages only in the female gonad, which indicates that these fatty acids could be a limiting factor for successful development of high quality eggs. The content of total monounsaturated fatty acids in male gonads, especially the fatty acid 16:1 n7, was significantly higher than in female gonads at the development and ripe stages. We also found differences between males and females in the use of some fatty acids in the digestive gland, especially at the spawned stage. Our results have future application in developing protocols for rearing of this pearl oyster in hatcheries. Incorporating dietary supplements containing docosapolyenoic fatty acids into diets of pearl oyster broodstock could be a practical way to improve their performance, which is crucial for enhancing the viability of larvae and spat.
Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 2012
Wild female Crassostrea corteziensis oyster (n = 245) were analyzed over one year to understand the main ecophysiological events associated to gonad development. Different indicators (mainly biochemical) were analyzed to infer: i) utilization and accumulation of energy reserves (e.g. neutral lipids, carbohydrates, proteins; vitellogenin), ii) membrane components provided by the diet as essential nutrients and indicative of cell proliferation (e.g. highly unsaturated fatty acids linked to phospholipids, sterols), iii) indicators of food availability (chlorophyll a in water, pigments in tissues, specific fatty acids and sterols), iv) gonad development (e.g. gonad coverage area, vitellin). A PCA analysis was applied to 269 measured variables. The first PC (PC1) was composed of total carbohydrate and lipid concentration, percentage of esterified sterols, fatty acids specific of diatoms; 16:1n−7/16:0, 20:5n−3 in neutral lipids with positive loadings and non methylene −interrupted fatty acids (NMI) in neutral lipids with negative loadings. The second PC (PC2) was composed of 18:4n−3 in lipid reserves and the concentration of zeaxanthin, a pigment typical of cyanobacteria with positive loadings and the proportion of 20:4n−6 in polar lipids with negative loading. The third PC (PC3) was composed of gonad coverage area (GCA) and the concentration of vitellin. Variation in GCA confirms that gonad development began in April with an extended period of spawning and rematuration from April to November. The PCA further shows that a second period of minimal maturation from November to March corresponds to the accumulation of reserves (PC1) together with an initial high availability of food (PC2) at the beginning of this period. These two periods are in accordance with the classical periods of allocation of energy to reserves followed by gonad development reported for several mollusks.
2012
Wild female Crassostrea corteziensis oyster (n = 245) were analyzed over one year to understand the main ecophysiological events associated to gonad development. Different indicators (mainly biochemical) were analyzed to infer: i) utilization and accumulation of energy reserves (e.g. neutral lipids, carbohydrates, proteins; vitellogenin), ii) membrane components provided by the diet as essential nutrients and indicative of cell proliferation (e.g. highly unsaturated fatty acids linked to phospholipids, sterols), iii) indicators of food availability (chlorophyll a in water, pigments in tissues, specific fatty acids and sterols), iv) gonad development (e.g. gonad coverage area, vitellin). A PCA analysis was applied to 269 measured variables. The first PC (PC1) was composed of total carbohydrate and lipid concentration, percentage of esterified sterols, fatty acids specific of diatoms; 16:1n−7/16:0, 20:5n−3 in neutral lipids with positive loadings and non methylene −interrupted fatty acids (NMI) in neutral lipids with negative loadings. The second PC (PC2) was composed of 18:4n−3 in lipid reserves and the concentration of zeaxanthin, a pigment typical of cyanobacteria with positive loadings and the proportion of 20:4n−6 in polar lipids with negative loading. The third PC (PC3) was composed of gonad coverage area (GCA) and the concentration of vitellin. Variation in GCA confirms that gonad development began in April with an extended period of spawning and rematuration from April to November. The PCA further shows that a second period of minimal maturation from November to March corresponds to the accumulation of reserves (PC1) together with an initial high availability of food (PC2) at the beginning of this period. These two periods are in accordance with the classical periods of allocation of energy to reserves followed by gonad development reported for several mollusks.
Aquaculture Research, 2008
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.
Fisheries Science, 2014
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 lg/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.
Cryobiology, 2006
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 eVective at predicting fertilizing ability. Techniques using Xuorescent 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 Xow cytometry and to assess viability of eggs by Xuorescence microscopy. The Xuorescent 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 diVerent 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 D 0.004) and thawing treatments (P D 0.04), and mitochondrial membrane potential (P D 0.0015) were correlated with motility. Fertilizing ability was correlated with cryoprotectant treatments (P D 0.0258) and with mitochondrial membrane potential (P D 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 D 0.002). Thawed eggs were not stained with FDA (<1%) and larvae were not produced.