Higher viscous solution induces smaller droplets for cell-enclosing capsules in a co-flowing stream

Mechanical strength of cell-enclosing capsules governs the success of the transplantation of enclosed cells in vivo for cell therapy. Mechanical strength closely correlates with the concentration and molecular weight of the polymers present in the aqueous solution that end up in the capsules, and the viscosity of the aqueous polymer solution also depends on these two factors. Three aqueous solutions differing in viscosity (1.0, 36, and 194 mPa s) were extruded from a needle (300 microm inner diameter) at a velocity of 1.2 cm/s into an ambient co-flowing liquid paraffin laminar stream. Smaller droplets were obtained from a higher viscous solution. At a liquid paraffin velocity of 23.5 cm/s, the diameter of droplets obtained from the highest viscous solution (194 mPa s)) was 44 +/- 4 microm, and it represented 40% and 20% of that from droplets in solutions of 36 and 1.0 mPa s viscosity, respectively. The cells enclosed in these droplets maintained more than 95% viability during the droplet breakup process independent of the viscosity of the aqueous solution (p > 0.50). In addition, retrieved cells from the droplets showed the same proliferation profiles as the cells that were not subjected to the droplet breakup process, on tissue culture dishes (p > 0.13).     

Development of mammalian cell-enclosing calcium-alginate hydrogel fibers in a co-flowing stream

A jetting technique in a liquid-liquid co-flowing stream was applied to the preparation of mammalian cell-enclosing calcium-alginate (Ca-alg) hydrogel fibers of several hundred micrometers in cross-sectional diameter. One percent alginate aqueous solution was extruded from needles (270, 480, 940 microm inner diameter) into a co-flowing laminar stream of 100 mM aqueous calcium chloride solution. The extruded alginate solution was stretched by the CaCl(2) solution, which is known as a "jetting process", and the Ca-alg hydrogel fibers were formed by gelation of the alginate solution through the uptake of calcium ions in the CaCl(2) solution. The cross-sectional diameter of the hydrogel fibers could be controlled from approximately 100-800 microm by changing the velocities of the alginate and CaCl(2) solution, and the inner diameter of the needle. Approximately 95% of bovine carotid artery vascular endothelial cells remained alive after the process of preparing hydrogel fibers in a co-flowing stream, demonstrating that the cell-enclosing process scarcely influences the viability of the enclosed cells.     

Production of cell-enclosing hollow-core agarose microcapsules via jetting in water-immiscible liquid paraffin and formation of embryoid body-like spherical tissues from mouse ES cells enclosed within these microcapsules

We developed agarose microcapsules with a single hollow core templated by alginate microparticles using a jet-technique. We extruded an agarose aqueous solution containing suspended alginate microparticles into a coflowing stream of liquid paraffin and controlled the diameter of the agarose microparticles by changing the flow rate of the liquid paraffin. Subsequent degradation of the inner alginate microparticles using alginate lyase resulted in the hollow-core structure. We successfully obtained agarose microcapsules with 20-50 microm of agarose gel layer thickness and hollow cores ranging in diameter from ca. 50 to 450 microm. Using alginate microparticles of ca. 150 microm in diameter and enclosing feline kidney cells, we were able to create cell-enclosing agarose microcapsules with a hollow core of ca. 150 microm in diameter. The cells in these microcapsules grew much faster than those in alginate microparticles. In addition, we enclosed mouse embryonic stem cells in agarose microcapsules. The embryonic stem cells began to self-aggregate in the core just after encapsulation, and subsequently grew and formed embryoid body-like spherical tissues in the hollow core of the microcapsules. These results show that our novel microcapsule production technique and the resultant microcapsules have potential for tissue engineering, cell therapy and biopharmaceutical applications.     

Agarose–gelatin conjugate for adherent cell-enclosing capsules

Spherical capsules were prepared by extruding aqueous agarose–gelation conjugate solution into co-flowing liquid paraffin at 38°C and cooling the resultant emulsion. Capsule diameter was controlled between 40 and 250 μm by changing the velocity of the liquid paraffin. Adherent Crandall–Reese feline kidney cells enclosed in conjugate capsules of 141 ± 23 μm diam. had a higher degree of proliferation than those in unmodified agarose capsules. Mitochondrial activity, detected for cell-enclosing conjugate capsules normalized against unit volume of gel, was about double that of unmodified agarose capsules over 28 days. These results demonstrated the feasibility of agarose–gelatin conjugate as a material of cell-enclosing capsules.     

Competing two enzymatic reactions realizing one‐step preparation of cell‐enclosing duplex microcapsules

The usefulness of cell‐enclosing microcapsules in biomedical and biopharmaceutical fields is widely recognized. In this study, we developed a method enabling the preparation of microcapsules with a liquid core in one step using two enzymatic reactions, both of which consume H₂O₂ competitively. The microcapsule membrane prepared in this study is composed of the hydrogel obtained from an alginate derivative possessing phenolic hydroxyl moieties (Alg‐Ph). The cell‐enclosing microcapsules with a hollow core were obtained by extruding an aqueous solution of Alg‐Ph containing horseradish peroxidase (HRP), catalase, and cells into a co‐flowing stream of liquid paraffin containing H₂O₂. Formation of the microcapsule membrane progressed from the surface of the droplets through HRP‐catalyzed cross‐linking of Ph moieties by consuming H₂O₂ supplied from the ambient liquid paraffin. A hollow core structure was induced by catalase‐catalyzed decomposition of H₂O₂ resulting in the center region being at an insufficient level of H₂O₂. The viability of HeLa cells was 93.1% immediately after encapsulation in the microcapsules with about 250 µm diameter obtained from an aqueous solution of 2.5% (w/v) Alg‐Ph, 100 units mL^?1 HRP, 9.1 × 10⁴ units mL^?1 catalase. The enclosed cells grew much faster than those in the microparticles with a solid core. In addition, the thickness of microcapsule membrane could be controlled by changing the concentrations of HRP and catalase in the range of 13–48 µm. The proposed method could be versatile for preparing the microcapsules from the other polymer derivatives of carboxymetylcellulose and gelatin. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1528–1534, 2013     

Electrostatic encapsulation and growth of plant cell cultures in alginate.

The growth of callus tissue from African Violets, encapsulated in alginate using electrostatics, was investigated as well as the mechanism of alginate droplet formation. Alginate microbeads as small as 500 (+/-50) microns in diameter could be produced by electrostatic extrusion directly from a plastic syringe (1900 micron extrusion orifice), in the absence of a needle. Video analysis of the mechanism of electrostatic alginate droplet formation from the syringe showed the development of a Taylor cone-like droplet which extended to form a thin strand that then broke up into droplets. Autoclaving of the alginate/medium solution significantly reduced its viscosity, giving smaller beads. Calculated microbead diameters agreed well with experimental values. Callus tissue from leaf explants was successfully immobilized and cultured using electrostatic extrusion. Tissue immobilized using 4% alginate in medium and cultured on agar grew best, producing a complete plantlet within four months. The long-term aim is to develop an effective method for large production of artificial seeds.     

Fabrication of artificial endothelialized tubes with predetermined three-dimensional configuration from flexible cell-enclosing alginate fibers

One possible strategy for creating three-dimensional (3D) tissue-engineered organs in vitro is to develop a vasculature for sufficient transport of oxygen and nutrients within these constructs. Here, we describe a novel technique to fabricate endothelialized tubes with predetermined 3D configuration, as a starting point for self-developing capillary-like networks in vitro. Calcium-alginate hydrogel fibers of ca. 250 and 500 mum in diameter, enclosing bovine carotid artery vascular endothelial cells (BECs), were used as templates for endothelialized tubes. Fibers were prepared by extruding a 2% (w/v) sodium alginate solution containing BECs into a 100 mM calcium chloride solution flowing in the same direction. Fibers were embedded in type I collagen gels and enzymatically degraded by alginate lyase, resulting in channels with predetermined 3D configuration filled with a BEC suspension. Cells attached to and covered the surfaces of the channels. Exposing the cells to medium containing basic fibroblast growth factor resulted in their migration into the ambient collagen gel and self-assembly into capillary-like structures. These results demonstrate that using artificial endothelialized tubes with predetermined 3D configuration, as a starting point for a self-developing capillary-like network, could be potentially useful for constructing 3D tissue-engineered organs.     

Immobilization of Saccharomyces uvarum cells in porous beads of polyacrylamide gel for ethanolic fermentation

Summary A procedure which does not involve the use of an immiscible organic solvent phase is described for the entrapment of yeast cells in porous beads of polyacrylamide gel. The cells are rapidly dispersed at 4° C in an aqueous solution containing sodium alginate and acrylamide-N,Nmethylene-bis-acrylamide monomer, and the suspension is immediately dropped into a solution of calcium formate to give calcium alginate coated beads. Polyacrylamide gel forms within the bead. The calcium alginate is subsequently leached out of the composite bead with either sodium citrate or potassium phosphate buffer solution. Cells of Saccharomyces uvarum ATCC 26 602 entrapped in such polyacrylamide beads ferment cane molasses in batch mode at higher specific ethanol productivity than a free cell suspension. Their volumetric productivity in continuous fermentation is higher than that of Ca²⁺-alginate immobilized cells.NCL Communication No. 4383     

Effect of ice nucleation by droplet of immobilized silver iodide on freezing of rabbit and bovine embryos

Silver iodide was immobilized by applying the insoluble reaction between sodium alginate and calcium chloride. The immobilized silver iodide was immersed into a freezing solution in order to trigger ice nucleation. Temperature change during cooling and postthaw in vitro development of embryos were examined in order to evaluate the effectiveness of the immobilized silver iodide (AgI alginate-gel droplet) on embryo development. Samples containing the AgI alginate-gel droplets released the latent heat of fusion at a higher subzero temperature than samples without the AgI alginate-gel droplets. When the AgI alginate-gel droplet was added to the freezing solution of rabbit and bovine embryos, they were successfully preserved in liquid nitrogen.     

Characterization of an encapsulation device for the production of monodisperse alginate beads for cell immobilization

An encapsulation device, designed on the basis of the laminar jet break-up technique, is characterized for cell immobilization with different types of alginate. The principle of operation of the completely sterilizable encapsulator, together with techniques for the continuous production of beads from 250 microm to 1 mm in diameter, with a size distribution below 5%, at a flow rate of 1-15 mL/min, is described. A modification of the device, to incorporate an electrostatic potential between the alginate droplets and an internal electrode, results in enhanced monodispersity with no adverse effects on cell viability. The maximum cell loading capacity of the beads strongly depends on the nozzle diameter as well as the cells used. For the yeast Phaffia rhodozyma, it is possible to generate 700 microm alginate beads with an initial cell concentration of 1 x 10(8) cells/mL of alginate whereas only 1 x 10(6) cells/ml could be entrapped within 400 microm beads. The alginate beads have been characterized with respect to mechanical resistance and size distribution immediately after production and as a function of storage conditions. The beads remain stable in the presence of acetic acid, hydrochloric acid, water, basic water, and sodium ions. The latter stability applies when the ratio of sodium: calcium ions is less than 1/5. Complexing agents such as sodium citrate result in the rapid solubilization of the beads due to calcium removal. The presence of cells does not affect the mechanical resistance of the beads. Finally, the mechanical resistance of alginate beads can be doubled by treatment with 5-10 kDa chitosan, resulting in reduced leaching of cells.     

Controlled Differentiation of Cells and Connective Tissue Fibers in Silver Staining

Controlled silver staining of connective tissue fibers and sometimes of these fibers and cells simultaneously can be obtained. 1. Fix in 10% formalin. Embed in paraffin and cut sections as usual, but do not mount them on slides. Deparaffinize and hydrate through xylene, alcohols and distilled water and henceforth treat them the same as frozen sections. Real frozen sections can also be used. 2. Treat with a freshly prepared 1% solution of KMnO₄, usually 15-60 sec, sometimes up to 10 min. 3. Wash in distilled water, 5-10 sec. 4. Decolorize in 2% potassium metabisulfite, 10-20 sec. 5. Place in distilled water, 1 min. 6. Sensitize with 2% iron alum, 1 min. 7. Place in distilled water, 1 min. 8. Impregnate in Gomori's silver oxide solution, 2 min. 9. Wash in a 1.5% aqueous solution of pyridine, about 15 sec. 10. Reduce in a mixture containing 0.25% gelatin and 2% formalin 1 min. 11. Repeat steps 7 to 10 once or several times until the connective tissue fibers are completely stained. For cell staining (which may fail) proceed as follows: After the first insufficient staining of the connective tissue fibers, rinse in distilled water, dip for 1 sec in Gomori's solution and reduce immediately in gelatin-formalin without previous washing in pyridined water. This step can be repeated. 12. If the staining is too strong, decolorize as needed in 2% iron alum. 13. Toning in 0.2% gold chloride, 5 min or more, followed by fixation in 5% sodium thiosulfate, 1 min, is optional. Counterstain as desired. 14. Wash in tap water, dehydrate, clear in xylene and mount in balsam. The same technique applied to sections attached to slides gives good results but inferior to that obtained in paraffin sections processed in the loose, unmounted condition.     

Rapid and onsite BOD sensing system using luminous bacterial cells-immobilized chip

A BOD monitoring system based on a bio-chip which immobilized luminous bacterium in micrometer-order holes were arrayed and fabricated by micro-machine techniques, was developed. The acrylic chip (3 cmx3 cm) comprises nine micro-holes (diameter: 700 microm or 1 mm, depth: 100 microm) arranged in a three by three array. Cells of the marine luminous bacterium, Photobacterium phosphoreum IFO 13896, which was grown at 15 degrees C for 15 h, were immobilized with 3% or 15% sodium alginate gel. BOD standard solutions or actual sample solution (approximately 10 microl) was fallen onto the cell-arrayed chip, and then the chip was incubated at 25 degrees C for 25 min. After incubation, bioluminescence from the each hole was gray-scaled and measured by a chemi-imager or newly developed onsite-type-monitoring system using a digital camera and a mobile-type personal computer. BOD values less than 16 ppm could detect by the chip, in particular, linear relationship at the concentrations between 0 and 16 ppm could be observed when luminous cells were immobilized with 3% sodium alginate gel. Steady bioluminescence was observed on the chip in the presence of BOD standard solution (GGA solution) which contained mineral elements. Furthermore, simultaneous detection of BOD values in various samples could be employed in the single chip. These results showed that the monitoring system with bio-chip could achieve high-through-put and onsite BOD detection. Our newly developed onsite-type BOD detection system which was used a digital camera and a (mobile) laptop computer was applied to measure and detect organic pollution due to biodegradable substances in wastewater treatment system. The same performance as the chemi-imager system was obtained for data of bioluminescence. The obtained BOD values showed a similar correlation with that of the conventional method for BOD determination (BOD5). These results suggested for successful achievement of high-though-put and onsite detection of BOD in practical.     

Effect of tank mixed adjuvants on the drift potential of phenmedipham formulations

The aim of this paper is to analyse the effect of adjuvants and formulations on drift. The spray liquids consisted of four adjuvants (Actirob 0.4 %, Tensiofix 0.2%, Breakthru 0.2%, Silwet L-77 0.1%) with water and with two formulations of Phenmediphame (C16H16N2O4, 4.45%): an emulsion-forming (EC) and a suspension concentrate (SC). A standard flat fan nozzle at a pressure of 3 bar was used. The droplet size spectrum of each combination was determined using a Malvern granulometer. The droplet size was characterized by the volume median diameter (VMD) and the percentage of spray volume contained in droplets <100 microm (%<100). The relative drift potential was measured for each combination of formulation and adjuvant in a wind-tunnel. This latter has a working section 2.0 m wide by 2.0 m high by 6.0 m long. The air-stream is drawn by a 1.2 m diameter axial flow fan, powered by a 22 kW electric motor. Wind speed was 5 m/s. Its uniformity was controlled by a three-dimensional sonic anemometer able to move on a linear translation beam placed in the tunnel cross section. The wind-tunnel was operated under ambient conditions and three repetitions were performed randomized in order to eliminate variations in temperature and humidity for each combination. The ground spray deposits were measured on glass fibber collectors using a fluorescent tracer dye (sodium fluorescein), at a concentration of 2.5 mg/l. The statistical analysis of the droplet spectrum showed that the Phenmediphame SC formulation generated droplets of higher size than the EC. The mean VMD values were respectively equal to 228+/-11 microm and 185+/-11 microm for these formulations. For SC formulation, Break-thru decreased the VMD while Tensiofix increased the %<100. This confirmed that the degree to which an adjuvant influences spray characteristics is very variable. The drift profiles produced by the different combinations were similar, but the relative drift potential was significantly different comparing SC and EC formulations: it respectively reached 0.8+/-0,08% and 1.2+/-0,08%, whatever the adjuvant used in the liquid. Clearly, when using a flat fan nozzle to spray Phenmediphame, the droplet size and the drift potential are mainly governed by the kind of formulation, even if an interaction between the formulation and the adjuvant exists.     

Effects of chain conformation and entanglement on the electrospinning of pure alginate

As a natural biopolymer, sodium alginate (SA) has been widely used in the biomedical field in the form of powder, liquid, gel, and compact solid, but not in the form of nanofiber. Electrospinning is an effective method to fabricate nanofibers. However, electrospinning of SA from its aqueous solution is still a challenge. In this study, an effort has been made to solve this problem and find the key reasons that hinder the electrospinning of alginate aqueous solution. Through this research, it was found that pure SA nanofibers could be fabricated successfully by introducing a strong polar cosolvent, glycerol, into the SA aqueous solutions. The study on the properties of the modified SA solution showed that increasing glycerol content increased the viscosity of the SA solution greatly and, meanwhile, decreased the surface tension and the conductivity of the SA solution. The rheological results indicated that the increase in glycerol content could result in the enhanced entanglements of SA chains. Two schematic molecular models were proposed to depict the change of SA chain conformation in aqueous solution with and without glycerol. The main contribution of glycerol to the electrospinning process is to improve the flexibility and entanglement of SA chains by disrupting the strong inter- and intramolecular hydrogen bondings among SA chains, then forming new hydrogen bondings with SA chains.     

Alginate hydrogel microspheres and microcapsules prepared by spinning disk atomization

Our spinning disk atomization (SDA) can, relative to other existing techniques, produce micron-sized particles with very narrow size distribution. The aim of this work is to present this technology for the production of alginate microspheres and microcapsules. We atomized and gelled aqueous alginate solutions into very narrowly dispersed microspheres with sizes ranging from 300 to 600 microm. Here, the interest is to produce, at high rate, particles of a given size with a narrow size distribution and also to show a new method of encapsulation using SDA. The viscosity and flow rate contributions in the drop formation is qualitatively analyzed to show how they affect droplet size. In addition, a technique for high degree of encapsulation is presented in which yeast is used as a model system. The production of yeast-loaded microspheres by SDA shows the potential of the technique for biotechnology applications.     

High performance liquid chromatographic determination of N-butyryl glucosamine in rat plasma

PURPOSE: A high performance liquid chromatography (HPLC) method for determination in plasma of N-butyryl glucosamine (GLBU), a highly water-soluble compound with no chromophore was developed. METHOD: To 100 muL of plasma containing GLBU was added fucose as internal standard. GLBU and fucose were derivatized using 1-phenyl-3-methyl-5-pyrazolone in the presence of sodium hydroxide at 70 degrees C for 30 min. The solution was neutralized with hydrochloric acid and the excess derivatizing reagent was extracted with chloroform. The aqueous layer was injected into an isocratic HPLC system consisting of an autoinjector, a single pump and a UV detector set at 245 nm. Two different 25 cm reversed phase columns were used, a 4 and a 10 microm C(18) columns. The mobile phase was a mixture of phosphate buffer (pH 7) and acetonitrile (80:20), which was run through a pump at a flow rate of 1.0 mL/min at ambient temperature. RESULTS: Derivatized fucose and GLBU appeared 24 and 28 min, and at 34 and 37 min using 4 and 10 microm columns, respectively. The assay was linear over the range of 0.2-200 microg/mL with a limit of quantification of 0.2 and 1 microg/mL for the 4 and 10 microm columns, respectively. The method was applied to the determination of GLBU in rat plasma after oral administration of 233 mg/kg of GLBU. CONCLUSION: The present assay is precise, and accurate with sufficient sensitivity for pharmacokinetic studies following therapeutically relevant doses.     

Dynamic changes in size distribution of emulsion droplets during ethyl acetate-based microencapsulation process

This study investigated the dynamic effect of the emulsification process on emulsion droplet size in manufacturing microspheres using ethyl acetate as an organic solvent. A dispersed phase consisting of poly(lactide-co-glycolide) and ethyl acetate was emulsified in a poly(vinyl alcohol) aqueous solution for a predetermined time ranging from 2 to 9, 16, 23, 30, 40, 50, or 60 minutes. Ethyl acetate was then quickly extracted to transform emulsion droplets into solidified microspheres, and their size distribution was determined. This experimental design allowed quantification of the size distribution of emulsion droplets over the course of emulsification. When emulsification time was extended from 2 to 60 minutes, the emulsion droplets decreased in size from 98.1 to 50.3 microm and their surface area increased from 0.07 to 0.29 m2/g. Overall, prolonging emulsification time up to 60 minutes resulted in the progressive evolution of smaller emulsion droplets (1-60 microm) and the simultaneous disappearance of larger ones (> 81 microm). Increases in the total number of microspheres and their surface area were caused mainly by continuous fragmentation of emulsion droplets before ethyl acetate extraction. The increase in the smaller microsphere population might also be due in part to shrinkage of microspheres. These results show that the onset of ethyl acetate extraction influenced the kinetics of the breakup and formation of emulsion droplets, thereby affecting to a great extent the size distribution of microspheres.     

Production and characterization of liquid-core capsules made from cross-linked acrylamide copolymers for biotechnological applications

A novel chemistry has been developed for the production of capsules composed of a hydrophobic liquid core surrounded by a cross-linked polyacrylamide/alginate membrane. These liquid-core capsules may be used in capsular perstraction for the removal of inhibitory products from bioprocesses and bioconversions. They have the advantage of having a high surface area to promote rapid mass transfer, while separation of the organic core phase from the aqueous environment by the capsule membrane prevents the formation of stable emulsions and potential problems associated with toxicity of the organic phase for microbial cells or enzymes. Monodisperse spherical liquid-core capsules of between 800 microm and 1.6 mm diameter, with high mechanical resistance, have been prepared by co-extrusion, using the jet break-up technique. Capsules produced from a solution of MBA/total monomer (5%) were found to be more elastic and have a higher burst force when exposed to chelating agents such as phosphate or citrate. The mechanical resistance was unaffected by buffer solutions in the pH range 4-9 and after sterilization at 121 degrees C for 20 min. Capsules having membranes composed of a copolymer of acrylamide and N-hydroxymethylacrylamide exhibited even higher mechanical stability toward chelating agents.     

Some properties of alginate gels derived from algal sodium alginate

Alginic acid in soluble sodium alginate turns to insoluble gel after contact with divalent metal ions, such as calcium ions. The sodium alginate character has an effect on the alginate gel properties. In order to prepare a suitable calcium alginate gel for use in seawater, the effects of sodium alginate viscosity and M/G ratio (the ratio of D-mannuronate to L-guluronate) on the gel strength were investigated. The wet tensile strengths of gel fibers derived from high viscosity sodium alginate were higher than those from low viscosity sodium alginate. The tensile strength increased with diminishing sodium alginate M/G ratio. Among the gel fibers tested, the gel fiber obtained from a sodium alginate I-5G (1% aqueous solution viscosity = 520 mPa·s, M/G ratio = 0.6) had the highest wet tensile strength. After 13 days treatment in seawater, the wet tensile strength of the gel fiber retained 36% of the original untreated gel strength. For sodium alginates with similar viscosities, the seawater tolerance of low M/G ratio alginate was greater than that of the high M/G ratio one. This study enables us to determine a suitable calcium alginate gel for use in seawater.     

Fine structure of the early stages of spermatogenesis in the Pacific oyster, Crassostrea gigas (Mollusca, Bivalvia)

The aim of this study is to describe the early stages of spermatogenesis of the Pacific oyster Crassostrea gigas using both light and electron microscopy. The gonad is formed by gonadal tubules invaginated in a connective tissue constituting a storage tissue. Myoepithelial cells surround each gonadal tubule and are associated with an acellular matrix delimiting the outer part of the tubule, the inner part is composed by intragonadal somatic cells associated with germinal lineage. Two types of spermatogonia are identified, where type I spermatogonia (Spg I) are large, scarce and pale cells leaned against the base of the tubule (nuclear diameter: 5.5+/-0.5 microm). Type II spermatogonia (Spg II) are clustered and dark cells which appear smaller than type I (nuclear diameter: 4.3+/-0.3 microm). The aspect of nuage-like material in cytoplasm is described from pale spermatogonia to primary spermatocytes (nuclear diameter: pachytene 3.6+/-0.3 microm, diplotene 3.4+/-0.3 microm), while no structure related to a chromatoid body was observed in oyster spermatocytes and spermatids.