Effect of chilling and cryopreservation on expression of Pax genes in zebrafish (Danio rerio) embryos and blastomeres

Cryopreservation is now common practice in the fields of aquaculture, conservation and biomedicine. However, there is a lack of information on the effect of chilling and cryopreservation at the molecular level. In the present study, we used real-time RT-PCR analysis to determine the effect of chilling and cryopreservation on expression of Pax2a, Pax2b, Pax5 and Pax8 which constitute one subgroup of the Pax gene family. As intact embryos of zebrafish have not yet been successfully cryopreserved, we have used two alternatives: chilling of intact embryos and cryopreservation of isolated blastomeres. Cryopreservation was found to affect the normal pattern of gene expression in zebrafish embryonic blastomeres. The trends, profile changes, in expression of Pax2a and Pax5 occurred to a lesser extent in frozen-thawed blastomeres than in fresh blastomeres whilst the opposite was true for Pax8. The trends in expression of Pax2b were delayed in frozen-thawed blastomeres compared to fresh blastomeres. Cryopreservation can therefore disrupt normal gene expression patterns in zebrafish embryonic blastomeres which could have a detrimental effect on embryo development.     

Effect of chilling on sox2, sox3 and sox19a gene expression in zebrafish (Danio rerio) embryos

Zebrafish embryos have not been cryopreserved due to their structural limitations. Although embryo survival rates have been used as the measured outcome for most of the cryopreservation protocols studied, there are very limited data available at the molecular level. This study focused on the effect of chilling and subsequent warming on gene expression of sox2, sox3 and sox19a which play vital roles in the development of zebrafish embryos. A quantitative RT-PCR approach was used to investigate gene expression following chilling at 0 °C for up to 180 min. The effect on gene expression was also studied during a 180 min warming period after chilling for 30 or 60 min. There were significant decreases in sox2 (up to 4-fold) and sox3 (up to 3-fold) expressions following chilling. Significant increases in gene expressions of sox2 (up to 2-fold), sox3 (up to 33-fold) and sox19a (up to 25-fold) were observed during warming in the embryos that had been chilled for 30 min. Similarly, significant increases were observed in sox2 (up to 3-fold) and sox3 (up to 2-fold) during warming in embryos that had been chilled for 60 min. These increases may be explained by compensation for the suppression observed during chilling and/or to activate repair mechanisms or maintain homeostasis.     

Extension of sperm motility leads to increased rates of fertilization and hatching in curimba,Prochilodus lineatus

The objective of this study was to evaluate the effects of six activating solutions on duration of sperm motility, fertilization rate (FR), and hatching rate (HR) of Prochilodus lineatus (Valenciennes, 1837). The activating solutions (SA) used were: SA₀ (199 mOsm kg^?1, pH 8.5), SA₁ (138 mOsm kg^?1, pH 7.5), SA₂ (256 mOsm kg^?1, pH 7.5), SA₃ (131 mOsm kg^?1, pH 10), NaCl (92 mOsm kg^?1, pH 7.5) and distilled water (32 mOsm kg^?1, pH 7.5). SA₁ induced the highest motility, FR and HR, compared with the other activating solutions. The lowest motility was obtained with SA₀, with no fertilization or hatching, whereas motility was zero with SA₂ and SA₃. It is possible to conclude that the solution SA₁ can be used for the activation of gametes during fertilization in induced reproduction of curimba to achieve higher fertilization and hatching rates. Thus, it was found that the osmolality and pH of activating solutions, probably with the participation of dissolved substances therein, are the main factors acting on semen motility after activation.     

Survivorship, development, and DNA damage in echinoderm embryos and larvae exposed to ultraviolet radiation (290-400 nm)

Laboratory experiments utilizing ecologically relevant irradiances of ultraviolet radiation (UVR) known to occur in shallow Gulf of Maine waters were conducted on the planktonic embryos and larvae of two common benthic echinoids; the green sea urchin Strongylocentrotus droebachiensis and the sand dollar Echinarachnius parma. Significant decreases in survivorship were observed in freshly fertilized embryos of both species with greater mortality in E. parma that was associated with the absence of UVR-absorbing compounds, the mycosporine-like amino acids. Experiments on blastula, gastrula, and prism larval stages of S. droebachiensis also showed significant decreases in survivorship, delays in development, and abnormal embryos and larvae associated with exposure to UVR. Additionally, all developmental stages of S. droebachiensis experimentally exposed to UVR resulted in significant increases in DNA damage, measured as cyclobutane pyrimidine dimer photoproducts. The observed delays in early cleavage and subsequent developmental stages for S. droebachiensis are correlated with DNA damage. It is postulated that cell cycle arrest at critical checkpoints after DNA damage, mediated by a suite of cell cycle genes, is a component of the observed UVR induced developmental delays.     

Effect of temperature on early development of white and lake sturgeon,Acipenser transmontanus and A. fulvescens

Synopsis The effect of constant incubation temperatures (between 10°C and 26°C) on the developmental rates was found to fit a similar exponential relationship in both the lake and white sturgeon embryos and larvae. Although the lake sturgeon had an overall slower rate of development than the white sturgeon, no statistically significant difference was detected in the slopes of the exponential equations describing the effect of temperature on developmental rate. The effect of these incubation temperatures on embryonic survival also did not differ between these two species. Both species exhibited optimal survival between 14–17° C and incipient mortalities occurred at 20°C. Temperatures above 20°C were lethal for white sturgeon embryos. No effect of low incubation temperature on survival was evident from this study. A comparison of these North American species with Eurasian acipenserids suggests that all the sturgeon that have been examined exhibit a similar influence of incubation temperature on developmental rate.     

Modulation of germination of embryos isolated from dormant and nondormant barley grains by manipulation of endogenous abscisic acid

Dormant and non-dormant barley (Hordeum distichum L.) grains with identical genetic backgrounds were obtained by maturing grains under different climate conditions. When isolated embryos from dormant grains were incubated in a well containing a fixed volume of water (300 l), the germination rate and percentage were dependent on the embryo number per well. A higher embryo number per well was correlated with a lower germination rate and percentage. However, this was not the case for the embryos isolated from nondormant grains. During germination, the endogenous cis-abscisic acid (ABA) in isolated embryos from both dormant and nondormant grains was analyzed. The inhibitory effect on germination of a higher number per well of isolated dormant embryos was due to diffusion of endogenous ABA out of the embryos and accumulation of ABA in the incubation medium. Moreover, there was de-novo synthesis of ABA in embryos isolated from dormant grains during incubation but not in embryos isolated from nondormant grains. The inhibitory effect of ABA on germination of embryos isolated from dormant grains could be mimicked by addition of ABA or the medium in which dormant embryos had been placed. Embryos isolated from nondormant grains were insensitive to addition of ABA and medium from dormant embryos. Our results demonstrate that diffusion of endogenous ABA, de-novo ABA synthesis and ABA sensitivity play a role in the control of germination. It is proposed that dormancy-breaking treatments act via changes to these processes.Abbreviations ABA cis-abscisic acid - E/W embryo(s) per well Prof. K.R. Libbenga (Institute of Molecular Plant Sciences, Leiden University) is thanked for fruitful discussions. B.V.D. was partly supported by E.E.C. BIOTECH program PL 920175.     

Reliable plantlet formation from embryos and seedling shoot tips of radiata pine

A method has been devised for the reliable production of plantlets from embryos and seedling shoot tips of Pinus radiata D.Don (radiata pine). Buds were induced on an agar or liquid Schenk and Hildebrandt (SH) medium containing 5.0 mg/l benzylaminopurine (BAP). Except for some abnormal buds, the buds grew into elongated shoots on an agar SH medium without cytokinin. The transfer of shoots from a SH medium to a Gresshoff and Doy (GD) medium was found to be an important pretreatment which increased the survival of the shoots when they were placed in a peat and pumice mix for root formation. Elongated shoots were induced to form roots under non-sterile conditions in a humid environment with occasional misting. An intervening 5-day treatment of shoots in an agar medium containing 2.0 mg/l indolebutyric acid (IBA) and 0.5 mg/l napthaleneacetic acid (NAA) significantly increased the percentage of shoots forming roots and the number of roots formed per shoot over control shoots placed directly in the peat:pumice mix. An enhanced level of CO₂ during root formation had no effect on the time of root formation or on the percentage of shoots forming roots. These results concerning the elongation, growth and rooting of adventitious shoots are now being applied to the development of very large numbers of plantlets starting from cotyledons from partially germinated seeds.     

Glycogen breakdown in cleaving Xenopus embryos is limited by ADP.

Xenopus eggs contain large stores of glycogen, but this glycogen is not glycolytically processed during cleavage. The Embden-Meyerhof pathway is inhibited by the absence of pyruvate kinase activity in vivo, and lactate and pyruvate are present at relatively low levels. In the late blastula, just preceding gastrulation, lactate levels increase, indicating the onset of glycogen breakdown and glycolytic flux. Glycolysis from microinjected [14C]glucose-6-phosphate could be transiently activated, however, by the coinjection of ADP into fertilized eggs, and constitutively activated by the injection of the ATPase potato apyrase, indicating the presence of all enzymes necessary for glycolytic activity. The isozyme profiles of pyruvate kinase and malic enzyme, two enzymes involved in carbon metabolism during cleavage or in the subsequent activation of glycogen breakdown, do not change between the egg and gastrula stages. These data suggest that the activation of glycogen breakdown and glycolysis in the late blastula is probably not a result of new gene activity but may be the metabolic consequence of increased free ADP that is then able to support the pyruvate kinase reaction.     

Large-scale in vitro embryo production and pregnancy rates from Bos taurus, Bos indicus, and indicus-taurus dairy cows using sexed sperm

Herein we describe a large-scale commercial program for in vitro production of embryos from dairy Bos taurus, Bos indicus, and indicus-taurus donors, using sexed sperm. From 5,407 OPU, we compared the number of recovered oocytes (n = 90,086), viable oocytes (n = 64,826), and embryos produced in vitro from Gir (Bos indicus, n = 617), Holstein (Bos taurus, n = 180), 1/4 Holstein × 3/4 Gir (n = 44), and 1/2 Holstein-Gir (n = 37) crossbred cows, and the pregnancy rate of recipient cows. Viable oocytes were in vitro matured (24 h at 38.8 °C, 5% CO₂ in air) and fertilized by incubating them for 18 to 20 h with frozen-thawed sexed sperm (X-chromosome bearing) from Gir (n = 8) or Holstein (n = 7) sires (2 × 10⁶ sperm/dose). Embryos were cultured in similar conditions of temperature and atmosphere as for IVM, with variable intervals of culture (between Days 2 and 5) completed in a portable incubator. All embryos were transferred fresh, after 24 to 72 h of transportation (up to 2,000 km). On average, 16.7 ± 6.3 oocytes (mean ± SEM) were obtained per OPU procedure and 72.0% were considered viable. Total and viable oocytes per OPU procedure were 17.1 ± 4.5 and 12.1 ± 3.9 for Gir cows, 11.4 ± 3.9 and 8.0 ± 2.7 for Holstein cows, 20.4 ± 5.8 and 16.8 ± 5.0 for 1/4 Holstein × 3/4 Gir, and 31.4 ± 5.6 and 24.3 ± 4.7 for 1/2 Holstein-Gir crossbred females (P < 0.01). The mean number of embryos produced by OPU/IVF and the pregnancy rates were 3.2 (12,243/ 3,778) and 40% for Gir cows, 2.1 (2,426/1,138) and 36% for Holstein cows, 3.9 (1,033/267) and 37% for 1/4 Holstein × 3/4 Gir, and 5.5 (1,222/224), and 37% for 1/2 Holstein-Gir. In conclusion, we compared oocyte yield from two levels of indicus-taurus breeds and demonstrated the efficiency of sexed sperm for in vitro embryo production. Culturing embryos during long distance transportation was successful, with potential for international movement of embryos.     

Modulation by ellagic acid of DCA-induced developmental toxicity in the zebrafish (Danio rerio)

The ability of ellagic acid (EA) to modulate dichloroacetic acid (DCA)-induced developmental toxicity and oxidative damage was examined in zebrafish embryos. Embryos were exposed to 20 mM EA administered concomitantly with 32 mM DCA at 4 hours postfertilization (hpf) and 20 h later. Embryos were observed through 144 hpf for developmental malformations, and production of superoxide anion (SA) and nitric oxide (NO) was determined in embryonic homogenates. DCA was shown to produce developmental abnormalities and significant levels of SA and NO in zebrafish embryos. EA exposure alleviated the developmental malformations observed in treated embryos and decreased the levels of SA and NO in those same embryos. Less than 10% of DCA + EA exposed embryos showed developmental malformations compared to 100% of embryos treated with DCA alone. Animals in this group that developed malformations were shown to have fewer defects than those treated with DCA only. Taken together, the results confirm the involvement of oxidative stress in the developmental toxicity of DCA in zebrafish embryos, and suggest possible protection against those effects with the use of antioxidants.