Comparative studies of sperm cryopreservation of diploid and tetraploid Pacific Oysters

This dissertation addressed comparative studies of sperm cryopreservation of diploid and tetraploid Pacific oysters, Crassostrea gigas, with an emphasis on the development of standardized and optimized protocols. This includes comparative ultrastructural differences between sperm from diploid and tetraploid oysters, methods for the rapid estimation of sperm concentration, optimization of cryopreservation, and evaluation of the mechanisms for sperm agglutination (formation of clumps or elongated noodles ) in thawed samples. Currently, cryopreserved sperm has not been commercialized in any aquatic species, and standardization and optimization could greatly benefit the potential commercialization of its use. In oysters specifically, cryopreserved sperm from tetraploids would facilitate the production of all-triploid seedstocks. In this study, sperm from tetraploid oysters were 25% larger in linear dimensions (lengths and widths), and 53% had 5 mitochondria compared to 4 in diploids. Spectrophotometric methods for rapid estimation of sperm concentration were developed and validated. The effects of cooling rate, single or combined cryoprotectants at various concentrations, equilibration time (exposure to cryoprotectant), straw size, and cooling method were evaluated for protocol optimization. Combination of the cryoprotectants polyethylene glycol (PEG; formula weight of 200) and methanol (for sperm from diploids) or PEG and propylene glycol (for sperm from tetraploids) were effective in retaining post-thaw motility only when PEG was at low concentrations (2-6%). Such effectiveness was especially manifested with sperm from tetraploids, for example, post-thaw motility as high as 50% was obtained with combined cryoprotectant. Sperm of tetraploid Pacific oysters were more susceptible to damage from cryopreservation procedures than were those of diploids, and male-to-male variation was significant for sperm from diploid and tetraploid oysters. Sperm agglutination was mainly due to the lack of sufficient cryoprotectant for specific sperm concentrations. These findings demonstrated the importance of standardization in sperm concentration and other procedures during cryopreservation. In addition, the systematic optimization of cryopreservation protocols involving interactions of multiple factors, recognition of male-to-male variation, and development of assays for sperm tolerance prior to freezing are all approaches important for the future potential commercialization of cryopreserved sperm in Pacific oysters and for other aquatic species as well

DYNAMIC VARIATION OF WATER QUALITY IN WEST LAKE AND MULTIVARIATE ANALYSIS OF ITS PRIMARY FACTORS

Dynamic variation of water quality in West Lake is analyzed based on the data of 1995. Principal Component Analysis is used to reveal the mutual relationships of various factors. It is shown that there exists an obvious special and temporal variation in the main factors of water quality. Annual values of TP, TN, and Chl.a fluctuate seasonally. In addition, Chl.a has a synchronous variation with water temperature, although being lagged a little, and closely relates to TP and TN. SD has a good negative relation with Chl.a. The results from Principal Component Analysis show that SD, Ec, Tw, pH and Chl.a are the most influential factors in water quality of the West Lake.Article信州大学理学部附属諏訪臨湖実験所報告 11: 21-27(1999)departmental bulletin pape

Neurodevelopmental toxicity assessments of alkyl phenanthrene and Dechlorane Plus co-exposure in zebrafish.

Alkyl phenanthrene (A-Phen) and Dechlorane Plus (DP) are ubiquitous environmental pollutants that widely co-exist in the environment. It has been established that both A-Phen and DP elicit neurotoxicity, but the potential interactive toxicity of these contaminants is not well-known. To determine whether a mixture of A-Phen and DP would exhibit interactive effects on neurodevelopment, we co-exposed 3-methylphenanthrene (3-MP), a representative of A-Phen, with DP. Our results illustrated that exposure to 5 or 20 μg/L 3-MP alone or in combination with 60 μg/L DP caused neurobehavioral anomalies in zebrafish. In accordance with the behavioral deficits, 3-MP alone or co-exposed with DP significantly decreased axonal growth of secondary motoneurons, altered intracellular Ca2+ homeostasis and induced cell apoptosis in the muscle of zebrafish. Additionally, 3-MP alone or co-exposed with DP significantly increased reactive oxygen species (ROS) and the mRNA levels of apoptosis-related genes. These findings indicate that 3-MP alone or co-exposed with DP induces neurobehavioral deficits through the combined effects on neuronal connectivity and muscle function. Chemical analysis revealed significant increases in 3-MP and DP bioaccumulation in zebrafish co-exposed with 3-MP and DP. Elevated bioaccumulation resulting from mixture exposure may represent a significant contribution of the synergistic effects observed in combined chemical exposure

Reproductive toxicity of low level bisphenol A exposures in a two-generation zebrafish assay: Evidence of male-specific effects

Bisphenol A (BPA), a high-volume chemical used to make polycarbonate plastic and epoxy resins, is a ubiquitous contaminant in environment and human body. To investigate the reproductive effects of long-term exposure to low concentrations of BPA, a two-generation study was conducted using the aquatic model species of zebrafish. Our findings revealed that exposure to 1 nM (0.228 μg/L) BPA for continuous two generations resulted in female-biased sex ratio in both F1 and F2 adult population, decreased sperm density, and decreased sperm quality as measured by motility, velocity, ATP content and lipid peroxidation in F1 and F2 males. Females were less sensitive to BPA exposures than males as no adverse effects were found in female gonads or gametes. Delayed hatching at 48 hpf and increased malformation and mortality were found in the offspring from BPA exposed F2, but not F1 parents. Most importantly, the adverse effect on larval development and survival from BPA exposed F2 parents was paternal-specific, resulting mainly from BPA exposed males. Subsequent transcription analysis of F2 male gonads revealed dysregulated mitochondrial biogenesis and significant activation of non-canonical Wnt/planar cell polarity and Wnt/Calcium signaling pathways. Gene expression analysis of larvae from BPA exposed F2 parents showed significant reduced expression of DNA methyltransferases such as dnmt1, dnmt3, and dnmt5. In conclusion, low level BPA exposures for continuous two generations not only affects sex ratio and sperm quantity/quality in F1 and F2 adults, reproductive success in offspring from F2 parents, but also perturbs various molecular pathways potentially contributing to these BPA induced male-specific reproductive defects.This is the publisher’s final pdf. The published article is copyrighted by Elsevier and can be found at: http://www.journals.elsevier.com/aquatic-toxicology/Keywords: Wnt signaling, BPA, Zebrafish, Chronic exposure, Sperm, Mitochondrial biogenesisKeywords: Wnt signaling, BPA, Zebrafish, Chronic exposure, Sperm, Mitochondrial biogenesi

Chronic PFOS exposures induce life stage-specific behavioral deficits in adult zebrafish and produce malformations in F1 offspring

Perfluorooctanesulphonicacid (PFOS) is an organic contaminant that is ubiquitous in the environment, wildlife, and humans. Few studies have assessed the effects of chronic PFOS exposure on central nervous system function in aquatic organisms. The present study defined the behavioral effects of varying life span chronic exposures to low dose PFOS in zebrafish. The zebrafish were treated with vehicle control or 0.5μM PFOS during 1-21, 21-120, or 1-120 day post fertilization (dpf). Chronic PFOS exposure impaired the adult zebrafish behavior mode under the tapping stimulus. The movement speed of 1-120 dpf exposed fish was significantly increased compared with control, while 1-21 and 21-120 dpf exposed groups were not severely affected. PFOS residues in F1 embryos derived from parental exposure during both the 1-120 and 21-120 dpf groups was significantly higher than control, and F1 embryos in these two groups showed obvious malformations, such as uninflated swim bladder (USB) and bent spine (BS). Larvae of the parental exposed to PFOS from 1-21 or 21-120 dpf elicited a higher swim rate than control in both the light and dark periods. Embryos derived from the 1-120 dpf group showed a statistically lower speed in the light period and a higher speed in the dark period as compared with control. Though there is little PFOS residue in 1-21 dpf group, the adverse behavioral effects on both adult and F1 larvae indicate that exposure during the first 21 dpf induce long-term neurobehavior toxicity. Our findings demonstrate that chronic exposure to low dose PFOS in different life stage adversely impacts adult behavior, subsequent offspring malformation, and larval behavior.This is an author's peer-reviewed final manuscript, as accepted by the publisher. The published article is copyrighted by Society of Environmental Toxicology and Chemistry and can be found at: http://onlinelibrary.wiley.com/journal/10.1002/%28ISSN%291552-8618.,Additional authors (Liu, Xiaojuan and Zhu, Guonian) appear and the author order is revised on the published version of this article.Keywords: perfluorooctanesulfonic acid, zebrafish embryo, chronic exposure, behavio

Early life perfluorooctanesulphonic acid (PFOS) exposure impairs zebrafish organogenesis

Additional authors (Zengxin Gai and Xue Ma) appear and the author order is revised on the published version of this article.As a persistent organic contaminant, perfluorooctanesulphonic acid (PFOS) has been widely detected in the environment, wildlife, and humans. The present study revealed that zebrafish embryos exposed to 16 µM PFOS during a sensitive window of 48-96 hour post-fertilization (hpf) disrupted larval morphology at 120 hpf. Malformed zebrafish larvae were characterized by uninflated swim bladder, less developed gut, and curved spine. Histological and ultrastructural examination of PFOS-exposed larvae showed structural alterations in swim bladder and gut. Whole genome microarray was used to identify the early transcripts dysregulated following exposure to 16 µM PFOS at 96 hpf. In total, 1,278 transcripts were significantly misexpressed (p < 0.05) and 211 genes were changed at least two-fold upon PFOS exposure in comparison to the vehicle exposed control group. A PFOS-induced network of perturbed transcripts relating to swim bladder and gut development revealed that misexpression of genes were involved in organogenesis. Taken together, early life stage exposure to PFOS perturbs various molecular pathways potentially resulting in observed defects in swim bladder and gut development.This is an author's peer-reviewed manuscript. The published article is copyrighted by Elsevier and can be found at: http://www.journals.elsevier.com/aquatic-toxicology/.Keywords: Perfluorooctanesulfonic acid, Zebrafish embryo, Developmental toxicity, Swim bladder, GutKeywords: Perfluorooctanesulfonic acid, Zebrafish embryo, Developmental toxicity, Swim bladder, Gu

Viability of an enzymatic mannitol method to predict sugarcane deterioration at factories

In the present study, a shape-independent differential scanning calorimeter (DSC) technique was used to measure the dehydration response during freezing of sperm cells from diploid and tetraploid Pacific oysters, Crassostrea gigas. This represents the first application of the DSC technique to sperm cells from nonmammalian species. Volumetric shrinkage during freezing of oyster sperm cell suspensions was obtained at cooling rates of 5 and 20 degrees C/min in the presence of extracellular ice and 8% (v/v) concentration of dimethyl sulfoxide (DMSO), a commonly used cryoprotective agent (CPA). Using previously published data, sperm cells from diploid oysters were modeled as a two-compartment ball-on-stick model with a ball 1.66 microm in diameter and a stick 41 microm in length and 0.14 microm wide. Similarly, sperm cells of tetraploid oysters were modeled with a ball 2.14 microm in diameter and a stick 53 microm in length and 0.17 microm wide. Sperm cells of both ploidy levels were assumed to have an osmotically inactive cell volume, Vb, of 0.6 Vo, where Vo is the isotonic (or initial) cell volume. By fitting a model of water transport to the experimentally obtained volumetric shrinkage data, the best-fit membrane permeability parameters (Lpg and ELp) were determined. The combined-best-fit membrane permeability parameters at 5 and 20 degrees C/min for haploid sperm cells (or cells from diploid Pacific oysters) in the absence of CPAs were: Lpg = 0.30 x 10(-15) m(3)/Ns (0.0017 microm/min-atm) and ELp = 41.0 kJ/mole (9.8 kcal/mole). The corresponding parameters in the presence of 8% DMSO were: Lpg[cpa] = 0.27 x 10(-15) m(3)/Ns (0.0015 microm/min-atm) and ELp[cpa] = 38.0 kJ/mole (9.1 kcal/mole). Similarly, the combined-best-fit membrane permeability parameters at 5 and 20 degrees C/min for diploid sperm cells (or cells from tetraploid Pacific oysters) in the absence of CPAs were: Lpg = 0.34 x 10(-15) m(3)/Ns (0.0019 microm/min-atm) and ELp = 29.7 kJ/mole (7.1 kcal/mole). The corresponding parameters in the presence of 8% DMSO were: Lpg[cpa] = 0.34 x 10(-15) m(3)/Ns (0.0019 microm/min-atm) and ELp[cpa] = 37.6 kJ/mole (9.0 kcal/mole). The parameters obtained in this study suggest that optimal rates of cooling for Pacific oyster sperm cells range from 40 to 70 degrees C/min. These theoretical cooling rates are in close conformity with empirically determined optimal rates of cooling sperm cells from Pacific oysters, C. gigas

Photoinduced RNA interference using DMNPE-caged 2\u27-deoxy-2\u27-fluoro substituted nucleic acids in vitro and in vivo

The consequences of UVB and UVA irradiation on hatch rate, mortality, and malformation were studied in embryonic zebrafish (Danio rerio). The use of zebrafish embryos has expanded from traditional developmental models to diverse studies, including many techniques utilizing light exposure. To characterize useful indicators of photodamage, the responses and threshold limits of UV radiation as a function of embryonic stage and fish source were evaluated. Significant differences in UVB susceptibility were observed in embryos at 3, 6-7, 12, and 24h post-fertilization (hpf), with the 1000-cell stage (3 hpf) having greatest tolerance to UVB. Embryos derived from zebrafish raised in outdoor ponds were more tolerant to UVB than were embryos from laboratory-raised fish. Combinations of UVB and UVA exposure were used to confirm the presence of a competent photorepair system in zebrafish that could return otherwise malformed embryos to a normal phenotype. Overall, embryonic zebrafish had large tolerances (LD(50) of 850 J/cm(2)) to UVA, confirming their suitability for photoactivation and photorepair studies