SEARCH · PubMed Health

Results for “protocol optimization”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 253 records · Page 14Linked to original sources

PubMed2006

A novel contact assay for testing genotoxicity of chemicals and whole sediments in zebrafish embryos.

Broad consensus exists that whole-sediment exposure protocols represent the most realistic scenario to simulate in situ exposure conditions. So far, however, several endpoints including genotoxicity in vertebrate-based systems could be tested only after transfer of particle-bound substances into the aqueous phase. The present study was carried out to develop a protocol for generating a suspension of single cells from sediment-exposed zebrafish embryos that is suitable for detecting particle-bound genotoxicity in the alkaline single cell gel electrophoresis (comet assay). In this solid-phase genotoxicity assay, a whole-body cell suspension derived from zebrafish embryos exposed to native (whole) sediments is assayed in the comet assay. Several chemical and mechanical isolation procedures were compared to optimize cell yield and minimize DNA damage by the method itself. If compared to collagenase isolation, mechanical cell dissociation gave less DNA damage; trypsinization resulted in similarly low DNA damage but significantly lower cell yield. In order to test the optimized protocol, effects of well-known genotoxicants (4-nitroquinoline-N-oxide, nitrofurantoin, hydrogen peroxide, benzo[a]pyrene) and of two sediments from the upper Rhine River (Germany) on zebrafish embryos were investigated. Results documented clear-cut genotoxicity for all four substances and for one of the two whole-sediment samples. An ultraviolet (UV) light exposure of whole embryos and primary cultures from embryos elucidated only minor effects for the whole embryos compared to the primary cells. Consequently, UV irradiation cannot be suggested as a positive control in intact zebrafish embryos. In conclusion, the newly developed sediment contact assay can be recommended for the detection of both single substances but also the bioavailable fraction of the total hazard potential of sediments.

Animals↗

PubMedDate not supplied

Analysis of the responses of human spermatozoa to A23187 employing a novel technique for assessing the acrosome reaction.

Protocols for the use of A23187 in assessing the ability of human spermatozoa to acrosome-react and exhibit sperm-oocyte fusion have been developed and the results compared in two independent cohorts of infertile patients. Both bioassays were found to depend upon such factors as the dose and formulation of A23187, the duration of exposure, and the amount and type of protein used to supplement the medium. An optimal protocol for the hamster oocyte penetration test comprised a 3-hour exposure to 1.25-2.5 microM ionophore and gave penetration rates of 93.2 +/- 3.2% (11.26 +/- 1.27 sperm/egg) for a group of 33 fertile donors compared with 63.0 +/- 5.4% (4.73 +/- 0.81 sperm/egg) for a cohort of 56 patients consulting for infertility (P < 0.001). Higher doses (5.0-10.0 microM) of A23187 caused an inhibition of sperm-oocyte fusion in association with a loss of motility, although the integrity of sperm plasma membrane did not appear to be compromised and high rates (approximately 80%) of acrosome reaction were observed. A protocol for assessing the ability of viable human spermatozoa to acrosome-react in response to A23187 was developed, employing a fluorescein-conjugated lectin in concert with the hypoosmotic swelling test, which gave values of 20.1 +/- 2.6% and 13.6 +/- 1.6% for groups of fertile donors (n = 29) and infertile patients (n = 32) respectively (P < 0.05). Although only acrosome-reacted spermatozoa were capable of fusing with zona-free hamster oocytes, there was no significant correlation between the proportion of acrosome-reacted cells and the levels of sperm-oocyte fusion observed in two independent groups of patients, indicating that these bioassays are measuring different aspects of human sperm function. These results have implications for the way in which the responses of human spermatozoa to ionophore treatment are quantified and interpreted.

Acrosome↗

PubMed2005

Osmotic shock: a mechanosensitive channel blocker can prevent release of cytoplasmic but not periplasmic proteins.

When over-expressed in the cytoplasm of Escherichia coli, carboxylesterase Est55 of Geobacillus stearothermophilus was found to be released from cells upon osmotic shock. Comparing two osmotic shock protocols showed that release of Est55 was abolished in the absence of mechanosensitive channel MscL by one method but not the other. The discrepancy extended to several previously reported cytoplasmic proteins released by osmotic shock, including: EF-Tu, thioredoxin, and DnaK in E. coli. Stepwise analyses of parameters between these two protocols revealed that the use of mechanical pipetting instead of gentle dilution of cells prior to exposure to hypotonic solution abolished the effect of MscL. Furthermore, while this phenomenon of release of certain cytoplasmic proteins was sustained in all three wild type strains of E. coli, presence of gadolinium was able to serve as an MscL channel blocker and prevented release of Est55 and EF-Tu in the process. An optimized protocol of osmotic shock was developed from this study to provide a more reliable assessment of location of proteins in E. coli. This method allowed release of authentic periplasmic MalE and beta-lactamase proteins comparable to that by EDTA-lysozyme treatment.

Biological Transport↗

PubMed2025

Nondestructive Larval Genotyping of Danio rerio for Mitochondrial and Nuclear DNA Genetics.

The rapid advancement of nuclear and mitochondrial genomic editing tools has created an urgent need for efficient, nonlethal larval genotyping methods in zebrafish (Danio rerio) research. This study optimizes and validates a nondestructive proteinase K digestion method for mitochondrial and nuclear DNA genotyping while characterizing its impact on larval survival and gene expression. Using optimized protocol parameters, we demonstrate successful amplification of different mitochondrial and nuclear genetic loci with consistently high sensitivity. Molecular validation through PCR, restriction fragment length polymorphism analysis, and Sanger sequencing confirmed the specificity and reliability of the extracted DNA. The method successfully detected C-to-T base edits in the mt-tl1 gene introduced using the FusX TALE Base editor system, demonstrating its applicability to gene editing studies. Both 48-well and optimized 96-well formats were used, enabling this approach to be deployed at scale. This optimized method enables researchers to correlate genotypes with phenotypes in longitudinal studies while maintaining specimen viability, particularly valuable for investigating early-onset mitochondrial diseases, and utilizes standard laboratory equipment and reagents, facilitating widespread adoption in zebrafish research while adhering to ethical principles in reducing animal mortality.

Animals↗

PubMed1998

Accuracy and 'range of uncertainty' of oscillometric blood pressure monitors around the upper arm and the wrist.

BACKGROUND: Although they have been marketed widely, few data about the diagnostic accuracy of blood pressure monitors are available. METHODS: Repeated measurements of blood pressures in 85 patients were performed in random sequence with two oscillometric blood pressure monitors around the upper arm (Visomat OZ2) and the wrist (Omron R3( and with a standard sphygmomanometer. The oscillometric blood pressure monitors were validated according to protocols of the British Hypertension Society (BHS) and the American Association for the Advancement of Medical Instrumentation (AAMI). Subsequently, sensitivity and specificity of these monitors for the diagnosis of hypertension or exclusion of the possibility of its presence in a general medical outpatient population were calculated. RESULTS: Sphygmomanometric readings exceeded oscillometric blood pressure measurements by 3.7+/-7.5/4.8+/-5.6 mmHg (systolic/diastolic) for the upper arm and 5.7+/-6.2/6.8+/-6.8 mmHg for the wrist. Deviations occurred in both directions and were higher for blood pressures in the hypertensive range. Oscillometric blood pressure measurements at the upper arm, but not at the wrist, satisfied validation criteria of BHS and AAMI protocols. Optimal sensitivity and specificity for the diagnosis of hypertension, defined as blood pressure > 140/90 mmHg with a standard sphygmomanometer, was achieved with blood pressure limits of 133/82 mmHg for the Visomat OZ and 131/80 mmHg for the Omron R3. CONCLUSIONS: Average sphygmomanometer values exceed oscillometrically measured blood pressure values but individual disagreements cannot be predicted. Measurements at the upper arm are more accurate than are those at the wrist according to the validation protocols of the BHS and AAMI. Additional appraisal of sensitivities and specificities and of a 'range of uncertainty' for the diagnosis of hypertension may allow better judgement of accuracy of individual oscillometric blood pressure measurements.

Adolescent↗

PubMed2004

Considerations when designing a clinical trial of haematopoietic stem cell transplantation for autoimmune disease.

The design and conduct of clinical trials of haematopoietic stem cell transplantation (HSCT) for autoimmune diseases requires investigators to address issues unique to this therapeutic approach and patient population. The proper composition of the protocol team is central to success. It is important to recognize that transplant physicians are no longer also the disease experts when transplanting patients with autoimmune diseases, and a close collaborative relationship between these groups early in the design stage must continue through the care of patients on trial to the assessment of toxicity and response. The early involvement of statisticians expert in clinical trial design and patient representatives are also vital to developing the optimal protocol. Each step in design and implementation requires particular consideration of the unique aspects of applying HSCT to autoimmune diseases. Some areas discussed are the role of disease and transplant databases in designing and analysing clinical trials, design options for early-phase trials, maintaining clinical equipoise, eligibility criteria, blinding, outcome measures and statistical analysis, and the composition and role of the data safety and monitoring boards. Although no blueprint for designing and conducting a trial of HSCT for autoimmune diseases can be laid out, the process should take into consideration the issues highlighted herein.

Autoimmune Diseases↗

PubMed2025

Development of a highly efficient protoplast regeneration and transfection protocol for enhancing CRISPR genome editing of Brassica carinata.

Brassica carinata is an important oil crop with significant potential for food and industrial production. The application of the CRISPR/Cas9 genome editing tool in B. carinata could accelerate its breeding cycle. However, no efficient DNA-free gene editing method currently exists for this species. Protoplast-based CRISPR editing presents a promising solution, though it is often challenging for many crop species. In this study, we investigated several critical factors influencing in vitro shoot regeneration, including genotype, sugar type, selection and combination of plant growth regulators (PGRs), and culture duration on different media throughout various stages of protoplast development. As a result, we developed a highly efficient, five-stage protoplast regeneration protocol for B. carinata based on specific stages of protoplast development. Key findings of this study include the requirement for high concentrations of NAA and 2,4-D in the initial medium (MI) for cell wall formation, while a lower auxin concentration relative to cytokinin was necessary for active cell division (MII). For callus growth and shoot induction, a high cytokinin-to-auxin ratio was essential (MIII), and an even higher cytokinin-to-auxin ratio was optimal for shoot regeneration (MIV). For shoot elongation, low levels of BAP and GA3 were sufficient (MV). Our results also demonstrated that the duration of culture on different media and maintaining appropriate osmotic pressure at the early stages were crucial for successful protoplast regeneration. With this optimized protocol, we achieved an average regeneration frequency of up to 64% and a transfection efficiency of 40% using the GFP marker gene. This efficient protoplast regeneration protocol is now being employed for genome editing in our lab and is expected to significantly enhance the application of the CRISPR system in both basic research and the genetic improvement of B. carinata over the long term.

Brassica carinata↗

PubMed2005

An improved axenic system for studying pre-infection development of the parasitic plant Orobanche ramosa.

BACKGROUND AND AIMS: Broomrapes (Orobanche spp.) are holoparasitic weeds that cause devastating losses in many economically important crops. The molecular mechanisms that control early stages of host infection in Orobanche are poorly understood, partly due to the lack of experimentally tractable in vitro systems that allow the efficient application of molecular tools. Here an improved axenic system for the analysis of pre-infection stages in O. ramosa in the absence of the host plant is described. METHODS: An optimized protocol for seed disinfection, based on formaldehyde, was developed. Orobanche ramosa seeds were conditioned in Petri dishes with filter paper, stimulated by addition of the synthetic strigol analogue GR24, and the percentage of germination as well as attachment-organ formation was determined. KEY RESULTS: Treatment of O. ramosa seeds with tobacco-root exudate or with GR24 resulted in highly reproducible germination rates around 70 %. A conditioning period of 8 d was both necessary and sufficient to allow optimal germination in response to GR24. Conditioned seeds that were dehydrated for several months remained fully responsive to GR24 without the need of a new conditioning period. Treatments as short as 5 min with GR24 were sufficient to fully and irreversibly induce the seed germination response. Approximately half of the germinated seeds initiated attachment-organ development. Similar rates of attachment organ induction were also detected in the rare cases of seeds that had germinated spontaneously on water. CONCLUSIONS: The results suggest that the conditioning period produces persistent changes in the seeds required for responsiveness to external stimulants. The rapid action of GR24 suggests that it may act via a receptor-mediated signalling mechanism. While germination in O. ramosa is induced by exogenous stimuli, attachment organ differentiation appears to be triggered by unknown endogenous signals. The new in vitro culture system will have useful applications for the molecular analysis of early stages of parasitic development in Orobanche.

Disinfectants↗

PubMed2001

An economical method for (15)N/(13)C isotopic labeling of proteins expressed in Pichia pastoris.

We report a new and cost-effective approach to prepare (15)N/(13)C labeled proteins for NMR using the Pichia pastoris expression system. Four protocols (P1 to P4) were defined and compared using recombinant Ovine interferon-tau (rOvIFN-tau). Our results demonstrate that in order to get full incorporation of (15)N and (13)C, the isotopes are not totally required during the initial growth phase of P. pastoris culture. The addition of small amounts of (15)N and (13)C compounds 6 h prior to the methanol induction phase is sufficient to obtain 99% incorporation of heavy isotopes into the protein. Our optimized protocol P4 is two-thirds less costly than the classical method using (15)N and (13)C isotopes during the entire growth phase.

Animals↗

PubMed2001

Photodynamic therapy for malignant mesothelioma: preclinical studies for optimization of treatment protocols.

Effective photodynamic therapy (PDT) depends on the optimization of factors such as drug dose, drug-light interval, fluence rate and total light dose (or fluence). In addition sufficient oxygen has to be present for the photochemical reaction to occur. Oxygen deficits may arise during PDT if the photochemical reaction consumes oxygen more rapidly than it can be replenished, and this could limit the efficacy of PDT. In this study we investigated the influence of the drug-light interval, illumination-fluence rate and total fluence on PDT efficacy for the photosensitizer meta-tetrahydroxyphenylchlorin (mTHPC). The effect of increasing the oxygenation status of tumors during PDT was also investigated. PDT response was assessed from tumor-growth delay and from cures for human malignant mesothelioma xenografts grown in nude mice. Tumor-bearing mice were injected intravenously with 0.15 or 0.3 mg.kg-1 mTHPC, and after intervals of 24-120 h, the subcutaneous tumors were illuminated with laser light (652 nm) at fluence rates of 20, 100 or 200 mW.cm-2. Tumor response was strongly dependent on the drug-light interval. Illumination at 24 h after photosensitization was always significantly more effective than illumination at 72 or 120 h. For a drug-light interval of 24 h the tumor response increased with total fluence, but for longer drug-light intervals even high total fluences failed to produce a significant delay in tumor regrowth. No fluence-rate dependence of PDT response was demonstrated in these studies. Nicotinamide injection and carbogen breathing significantly increased tumor oxygenation and increased the tumor response for PDT schedules with illumination at 24 h after photosensitizer injection.

Animals↗

PubMed1995

In vitro study of the catecholaminergic metabolism of locus coeruleus neurones by differential normal pulse voltammetry.

The aim of the present work was to measure, by voltammetry, the catecholaminergic metabolic activity of rat locus coeruleus (LC) neurones in brain slices. For this new experimental approach, we used an optimized protocol of slice preparation intended to prevent neuronal damages due to brain ischaemia. Our results show that the LC neurones exhibit in vitro a stable spontaneous catecholaminergic metabolic activity and that, as in vivo, 3,4-dihydroxyphenylacetic acid (DOPAC) is likely to be the main contributor to the recorded signal. This catecholaminergic metabolic activity can be pharmacologically altered by administering carbachol and clonidine to the superfusion fluid. We also determined the values of bath temperature and superfusion flow rate providing, in our methodological conditions, an optimal catecholaminergic metabolic activity. Finally, we took advantage of both the direct accessibility to the LC and the compactness of this nucleus to determine the spatial resolution of differential normal pulse voltammetry. In conclusion, the study of the subregional mechanisms controling the catecholaminergic metabolism in LC neurones can be performed in brain slices by differential normal pulse voltammetry.

3,4-Dihydroxyphenylacetic Acid↗

PubMed1997

Optimization of the isolation and effective use of mRNA from rat mast cells.

To define the molecular regulation of mast cell phenotype and function optimized procedures must be available to study mRNA from mast cells freshly isolated from tissues. However, rat peritoneal mast cells (PMC) contain large amounts of the proteoglycan heparin, and unfortunately, this molecule which is a potent inhibitor of reverse transcriptase (RT) and Taq polymerase and thus RT-PCR, copurifies with RNA. Here we describe an optimized protocol for extracting and amplifying RNA from rat PMC. Mast cells were isolated from rat peritoneum and a method modified from that of Chomczynski and Sacchi (1987) was used to extract the RNA. Following the removal of heparin by heparinase digestion, first strand cDNA synthesis was primed with oligo-dT and the resulting cDNA was quantified by rapid paper chromatography. The use of a detection system for the reverse transcription reaction ensured that the production of cDNA had occurred and allowed subsequent PCR testing to be optimal. cDNA thus produced can be used to detect relatively specific (histidine decarboxylase) and non-specific (beta-actin) mast cell products. Our PCR studies have shown a 300-fold increase in sensitivity over RNA processed by other methods.

Animals↗

PubMedDate not supplied

Simple protocols to determine dust potentials from cattle feedlot soil and surface samples.

Cattle feedlot dust is an annoyance and may be a route for nutrient transport, odor emission, and pathogen dispersion, but important environmental factors that contribute to dust emissions are poorly characterized. A general protocol was devised to test feedlot samples for their ability to produce dust under a variety of environmental conditions. A blender was modified to produce dust from a variety of dried feedlot surface and soil samples and collect airborne particles on glass fiber filters by vacuum collection. A general blending protocol optimized for sample volume (150-175 cm3), blending time (5 min of pre-blending), and dust collection time (15 s) provided consistent dust measurements for all samples tested. The procedure performed well on samples that varied in organic matter content, but was restricted to samples containing less than 200 to 700 g H2O kg(-1) dry matter (DM). When applied to field samples, the technique demonstrated considerable spatial variability between feedlot pen sites. Mechanistically, dust potential was related to moisture and organic matter content. An alternative protocol also demonstrated differences within pen sites in maximum dust potential and dust airborne residence time. The two protocols were not intended, nor are they suitable, for predicting actual particulate matter emissions from agricultural sources. Rather, the protocols rapidly and inexpensively compared the potential for dust emission from samples of differing composition under a variety of environmental conditions.

Agriculture↗

PubMed1998

Rapid method for fluorescent in situ ribosomal RNA labelling of Cryptosporidium parvum.

A method for fluorescence in situ hybridization (FISH) is described that requires less than 1 h duration. Oocysts were resuspended in 50% ethanol and incubated at 80 degrees C for 10 min for simultaneous fixation and permeabilization. Samples were than incubated with the oligonucleotide probe at 48 degrees C for more than 30 min. The rRNA binding specificity of the optimized protocol was confirmed. FISH was found to be valuable as a second label for oocysts presumptively identified immunofluorescently, but required more than an order of magnitude signal amplification for independent use. The number of oligonucleotide probes bound per oocyst was compared with the copy number of 18S rRNA molecules per oocyst to provide a measure of the labelling efficiency of the FISH method. Hybridization kinetics were also analysed. These data indicate that significant further increases in the brightness of FISH-labelled oocysts cannot be achieved by further optimization of the pre-treatment and hybridization conditions.

Animals↗

PubMed1998

Continuous tracheal gas insufflation enables a volume reduction strategy in hyaline membrane disease: technical aspects and clinical results.

OBJECTIVE: Instrumental dead space wash-out can be used to improve carbon dioxide clearance. The aim of this study was to define, using a bench test, an optimal protocol for long-term use, and to assess the efficacy of this technique in neonates. DESIGN: A bench test with an artificial lung model, and an observational prospective study. Dead space wash-out was performed by continuous tracheal gas insufflation (CTGI), via six capillaries molded in the wall of a specially designed endotracheal tube, in 30 preterm neonates with hyaline membrane disease. SETTING: Neonatal intensive care unit of a regional hospital. RESULTS: The bench test study showed that a CTGI flow of 0.5 l/ min had the optimal efficacy-to-side-effect ratio, resulting in a maximal or submaximal efficacy (93 to 100%) without a marked increase in tracheal and CTGI circuit pressures. In the 30 newborns, 15 min of CTGI induced a significant fall in arterial carbon dioxide tension (PaCO2), from 45 +/- 7 to 35 +/- 5 mmHg (p = 0.0001), and in 14 patients allowed a reduction in the gradient between Peack inspirating pressure and positive end-expiratory pressure from 20.8 +/- 4.6 to 14.4 +/- 3.7 cmH2O (p < 0.0001) while keeping the transcutaneous partial pressure of carbon dioxide constant. As predicted by the bench test, the decrease in PaCO2 induced by CTGI correlated well with PaCO2 values before CTGI (r = 0.58, p < 0.002) and with instrumental dead space-to-tidal volume ratio (r = 0.54, p < 0.005). CONCLUSION: CTGI may be a useful adjunct to conventional ventilation in preterm neonates with respiratory disease, enabling an increase in CO2 clearance or a reduction in ventilatory pressure.

Artificial Organs↗

PubMed2001

An immunoblotting method for high-resolution isoelectric focusing of protein isoforms on immobilized pH gradients.

Post-translational modifications such as phosphorylation and acetylation are important elements for regulating the activity of enzymes or structural proteins. These modifications give rise to isoforms that are often not resolved by separation methods relying on the size of proteins. Here, we optimized an isoelectric focusing (IEF)-immunoblotting method suitable for analyzing protein isoforms in total cell extracts. The separations were carried out in parallel on commercially available immobilized pH gradient slab gels (IPG). The buffer used for separation contained urea, thiourea, dithiothreitol, as well as the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate (CHAPS), and was designed to match those used in two-dimensional polyacrylamide gel electrophoresis (PAGE) separations where efficient solubilization is required. Proteins were transferred to membranes by passive diffusion in the presence of 4 M guanidinium chloride using protocols optimized for several protein classes (tubulin, stathmin, 14-3-3 proteins) some of which required removal of CHAPS prior to transfer. In conjunction with narrow-range pH gradient gels, excellent resolution of isoforms differing by phosphorylation or acetylation was achieved. The usefulness of pI and titration curve calculations for predicting the pI shifts expected for post-translational modifications of proteins with known amino acid composition was demonstrated. Using stathmin--which contains four phosphorylation sites--as an example, the effects on the pI-shifts were well predicted. This sensitive and widely applicable IEF-blotting technology is expected to be especially suited for analyzing protein isoforms first detected by two-dimensional electrophoresis.

14-3-3 Proteins↗

PubMed1987

[Principles of the simultaneous radiation and polychemotherapy in advanced head and neck carcinoma].

Concomitant application of chemotherapeutic agents and radiotherapy increases the tumoricidal effects. In this report the biochemical, cell-kinetic and radiobiological interactions of the simultaneous radio-polychemotherapy are discussed. Systemic chemotherapy consisted of cis-dichlorodiammineplatinum (II) (cis-DDP; 60 mg/sqm), 5-fluorouracil (5-FU; 350 mg/sqm) and folinic acid (FA; 50 mg/sqm) on day 2 and 5-FU 350 mg/sqm/24 hrs and FA 100 mg/sqm/24 hrs on days 2-5. Radiotherapy was applied in 13 fractions of 1.8 Gy delivered twice daily from days 3-11. The regimen was repeated on days 22 and 44, reaching the final dose of 70.2 Gy in 8 weeks. With this protocol, optimal tumor regression was achieved in very advanced squamous cell carcinoma of the head and neck. Survival rate after 2 years was 76.8% with 28/32 complete and 4/32 partial remissions. Using an intensive adjuvant treatment, the overall toxicity was tolerable allowing the application of the full therapeutic dosage in 95% without any interruptions.

Antineoplastic Combined Chemotherapy Protocols↗

PubMed2005

Successful cryopreservation of Pacific oyster (Crassostrea gigas) oocytes.

Protocols for cryopreservation of sperm and oocytes would provide the ultimate control over parental crosses in selective breeding programmes. Sperm freezing is routine for many species, but oocyte freezing remains problematic, with virtually zero success in aquatic species to date. This paper describes the development of a successful protocol for cryopreserving high concentrations of Pacific oyster (Crassostrea gigas) oocytes. Ethylene glycol (10%) and dimethyl sulfoxide (15%) were found to be the most effective cryoprotectants resulting in post-thaw fertilization rates of 51.0+/-8.0 and 45.1+/-8.3%, respectively. Propylene glycol was less effective and methanol resulted in zero fertilization post-thaw. The use of Milli-Q water rather than seawater as a base medium significantly improved fertilization (20.4+/-3.0 and 8.7+/-2.2%, respectively) as did the inclusion of a 5 min isothermal hold at -10 or -12 degrees C (35.9+/-5.0 and 31.9+/-4.6%, respectively). The optimal cooling rate post-hold was 0.3 degrees C min(-1), with virtually zero post-thaw fertilization with cooling rates of 3 and 6 degrees C min(-1). Using an optimized protocol, post-thaw fertilization rates for oocytes from eight individual females ranged from 0.8 to 74.5% and D-larval yields from 0.1 to 30.1%. For three individuals, larvae were reared through to spat. Development of D-larvae to eyed larvae and spat was similar for larvae produced from unfrozen (24.8+/-4.1% developed to eyed larvae and 16.5+/-3.2% to spat) and cryopreserved (28.4+/-0.6 and 18.7+/-0.5%, respectively) oocytes. The ability to cryopreserve large quantities of oyster oocytes represents a major advance in cryobiology and selective breeding.

Animals↗