Rearing medium dictates variability across replicates in untreated and arsenic challenged zebrafish larvae

ABSTRACTReproducibility and consistency are hallmarks of scientific integrity. Biological systems are inherently noisy, posing a challenge to reproducibility. This is particularly relevant to the field of environmental toxicology, where many unaccounted experimental parameters can have a marked influence on the biological response to exposure. Here, we extend the use of zebrafish as a robust toxicological model for studying the effects of inorganic arsenic (iAs) on liver biology. We observed that iAs toxicity in this system is not influenced by important parameters including genetic background, rearing container material or rearing volume but the dose response to iAs is influenced by the rearing medium. We compared mortality as a measure of iAs toxicity to embryos cultured in two standard rearing media: egg water made from dehydrated ocean salts dissolved in water and a defined embryo medium which is a pH adjusted, buffered salt solution. Larvae reared in egg water were more susceptible to iAs compared to those reared in embryo medium. This effect was independent of the pH differences between these solutions. These culture conditions did not cause any difference in the global hepatic transcriptome of control zebrafish. Further, no difference in the expression of genes involved in the unfolded protein response (UPR) in larvae exposed to iAs treatment or in a stress independent system to activate UPR genes by transgenic overexpression of activating transcription factor 6 (nAtf6) in hepatocytes was observed. However, the clutch-to-clutch variation in gene expression was significantly greater in larvae reared in egg water compared to those in embryo medium. These data demonstrate that egg water affects reproducibility across replicates in terms of gene expression and exacerbates iAs mediated toxic response. This highlights the importance of rigorous evaluation of experimental conditions to assure reproducibility.

Production and Characterization of Mouse Diploid Parthenogenetic Blastocyst Developed in Phosphate-Free Medium

Parthenogenesis is an artificial oocytes activation process without paternal contribution. Blastocyst, derived from parthenogenesis, is one of potential source for pluripotent stem cell propagation. Unfortunately, previous studies reported that parthenogenetic embryo did not achieve exhilarating blastocyst rate. One of the component that predicted inhibit parthenogenetic embryo development is phosphate. Therefore, we try to modify culture medium in order to overcome that problem. The aim of this research was to produce and analyze the characteristics of parthenogenetic blastocyst developed in phosphate-free medium. Mouse oocytes obtained from adult female DDY by superovulation. The activator was strontium chloride 10 mM and diploidization with cytochalasin B 5 μg/ml. Medium for activation and culture medium were modified rat 1 cell embryo medium (MR1ECM) which is phosphate free. The results showed that parthenotes that were cultured in phosphate free medium reached higher blastocyst rate compared to the other groups. The increase of phosphate in culture medium lead to impaired parthenogenetic embryos development. Further experiment was made to analyze the differences between fertilized and parthenogenetic embryo in this medium. The experiment showed that diploid parthenogenetic could achieve high blastocyst rate (30.9±1.3%). The quality of diploid parthenogenetic blastocyst, based on cells number, viability, and ICM ratio, was lower than fertilized blastocyst.

Equine blastocyst production under different incubation temperatures and different CO2 concentrations during early cleavage

Some basic parameters for equine invitro embryo production have not yet been established, including the optimum temperature for maturation and embryo culture, and the optimum CO2 concentration and pH during early embryo development. To explore this, we first performed cultures in incubators set at 37.2°C, 37.7°C or 38.2°C. At these temperatures, the corresponding maturation rates were 33%, 38% and 42%; cleavage rates were 84%, 86% and 88%; and blastocyst rates were 35%, 44% and 44% per injected oocyte. These rates did not differ significantly (P>0.2). We then evaluated three different CO2 concentrations (6%, 6.5% or 7% CO2) in 5% O2 for culture over Days 0–5 after intracytoplasmic sperm injection, using a commercial human embryo medium with added serum, at 38.2°C. The pH values of these media were 7.36, 7.33 and 7.29 respectively. In the presence of 6%, 6.5% or 7% CO2, cleavage rates were 68%, 80% and 70% respectively, and blastocyst rates per injected oocyte were 42%, 54% and 27% respectively. The blastocyst rate for the 7% CO2 treatment was significantly lower than that for the 6.5% CO2 treatment (P<0.05). We conclude that equine invitro embryo production is equally effective within the range of 37.2–38.2°C, but that equine early cleavage stage development is sensitive to small changes in CO2 atmosphere and/or medium pH.

177 IN VITRO MATURATION AND FERTILIZATION OF OVARIAN OOCYTES OF FREE-RANGING GEMSBOK (ORYX GAZELLA)

The gemsbok is a large antelope native to arid regions of southern Africa. Listed by the International Union for Conservation of Nature as a species of least concern, the gemsbok is an excellent model for the development of assisted reproductive techniques for the closely related but critically endangered scimitar-horned oryx (Oryx dammah) and addax (Addax nasomaculatus). Gemsbok were introduced to the White Sands Missile Range by the New Mexico Department of Game and Fish to preserve the habitat by providing big game hunting, which ensures the support and lobby of hunters. Gonads were collected from hunted gemsbok and transported in PBS to the laboratory, where gametes were harvested. Testes were stored at 4°C in PBS until sperm were needed for IVF (18 to 28 h). Ovaries were sliced to release follicular oocytes, which were placed in maturation medium (TCM-199 with FCS, pyruvate, gentamicin and LH, FSH and oestradiol) for 20- to 22-h in vitro maturation culture at 38.8°C in 5% CO2 in air. Oocytes were then washed [Tyrode lactate (TL)-HEPES with BSA, pyruvate and gentamicin] and placed in groups of 5 to 10 in 50-μL drops of IVF-TL medium supplemented with pyruvate, gentamicin and BSA. Sperm were allowed to swim out of sliced epididymides into room temperature IVF-TL medium and then equilibrated for 30 min at 38.8°C. Approximately 2 × 103 motile sperm were added to each oocyte drop and incubated under oil for 21 to 23 h at 38.8°C in 5% CO2 in air. Domestic cattle oocytes were matured in maturation medium at 39°C during shipment to the field site, where they were washed and transferred to IVF medium as described for gemsbok oocytes. Approximately 1.7 × 103 motile gemsbok sperm were added to each drop and oocytes were incubated as described for gemsbok oocytes. At the end of IVF culture, oocytes of both species were stripped of granulosa cells and placed in embryo medium (SOF supplemented with BSA, essential and nonessential amino acids, pyruvate and gentamicin; all media formulations from Applied Reproductive Technologies, Madison, WI, USA) at 38.8°C in 5% CO2 in air. Embryo culture medium was refreshed after 48 h. Embryos were removed from culture after 6 days, examined for cleavage and fixed in PBS:formalin for staining and further analysis. Although gemsbok sperm were capable of fertilizing domestic cow oocytes at the same rate (38.3%) as gemsbok oocytes (37.1%), the antelope oocytes cleaved at a higher rate (29% vs cattle at 15.3%). These results indicate that chilled epididymal gemsbok sperm is capable of fertilizing gemsbok and domestic cattle oocytes and that protocols designed for in vitro maturation and fertilization of cattle oocytes may be successfully used in the field to produce gemsbok embryos (Table 1). Table 1.Fertilization of in vitro-matured gemsbok and cattle oocytes by chilled epididymal gemsbok sperm The authors thank the New Mexico Department of Game and Fish, Troylyn Zimmerly, Dana Powers and the Biology Department of New Mexico Institute of Mining and Technology.