Renovation of a Drop Embryo Cultures System by Using Refined Mineral oil and the Effect of Glucose and/or Hemoglobin Added to a Serum-free Medium

Abstract

ABSTRACT. This study was conducted to evaluate whether refining mineral oil and the addition of hemoglobin and/or glucose to a serumfree medium could improve in vitro-development of embryos cultured in a chemicallysemi-defined microdroplet culture system. Block strain, outbred (ICR) mouse 1-or 2-cell embryos were cultured in 5 µl droplets of Chatot, Ziomek and Bavister medium overlaid with mineral oil of different types, and preimplantation development to the blastocyst stage was subsequently monitored. In the experiment 1, either Sigma (M-8410) or BDH (GPR TM ) mineral oil with or without washing was used for embryo culture and, distilled water (DW) or culture medium was used as a washing agent. As results, better (P<0.0001) development of 1-cell embryos was found in the Sigma than in the BDH; more blastocysts developed in Sigma oil washed with culture medium than in the others (37% vs. 0%). Subsequent ly, 1-(experiment 2) or 2-cell (experiment 3) embryos were cultured in the droplets overlaid with medium-washed Sigma oil, to which 0.001 mg/ml hemoglobin and/or 5.6 mM glucose were supplemented at the 1-cell and the 4-cell stages, respectively. Regardless of embryo stages, blastocyst formation was significantly improved by the addition of hemoglobin (54 to 48% vs. 42 to 31% in 1-cell and 83 to 78% vs. 65 to 68% in 2-cell embryos) and this effect was independent of glucose addition. In conclusion, the selection and washing of mineral oil, and the addition of hemoglobin is beneficial for improving the efficacy of a drop embryo culture system using a serum-free medium. KEY WORDS: glucose, hemoglobin, mineral oil, mouse embryo, preimplantation development.J. Vet. Med. Sci. 66(1): 63-66, 2004 Technical advance in gamete manipulation is fundamental for developing novel medical biotechnologies. Lots of information has been obtained from various model studies and we have developed the mouse model system of embryo culture employing serum-free media and outbred strain (Institute of Cancer Research; ICR) [10,11]. Subsequently, a culture technique using small microdroplet of a serumfree medium has been developed. In this system, commercially-purchased mineral oil is used for preparing microdroplets, so culture medium was continuously exposes to overlaid oil throughout the culture. It is possible that the artifacts included in overaid oil directly affect on the development of embryos cultured in microdroplet by continuous exposure during in vitro-culture. This hypothesis was confirmed by recent finding showing the influence of oil on embryo development during in vitro-culture [12]. In addition, a Chatot, Ziomek and Bavister (CZB) medium [1-4] was employed as a basic medium of our culture system, but this does not contain several substrates having embryotrophic action reported in previous studies [8][9][10][11].Consequently, a randomized, prospective study was designed to further improve our embryo culture system using the microdroplet of a serum-free medium. This study was conducted to examine 1) the effects of two types ...

Sterile filtered paraffin oil supports in vitro developmental competence in bovine embryos comparable to co-culture

“…The role of oil in embryo development has been demonstrated in many species including mice (4,(11)(12), pigs (14) and cows (9,13). Oil overlaying also helps to protect the embryos by neutralizing the toxic artifacts in culture systems (4).…”

“…The role of oil in embryo development has been demonstrated in many species including mice (4,(11)(12), pigs (14) and cows (9,13). Oil overlaying also helps to protect the embryos by neutralizing the toxic artifacts in culture systems (4). On the contrary, oil also has negative effects on embryo culture as it might reduce the full maturation potential of pig oocytes during in vitro meiotic maturation by interacting with steroid hormones, such as oestradiol and progesterone, in the medium (14).…”

“…Researchers have optimized culture systems for various embryo stages and species (4)(5)(6), thus advancing in vitro culture technology for blastocyst development. The most commonly used culture environment has been the micro drop culture method using oil rather than large scale medium, with the exception of certain specific culture methods.…”

“…Oil overlaying protects against dehydration and hydrogen concentration variation in the in vitro medium environment. Several studies investigating the effect of oil quality on embryo development (4,(8)(9)(10)(11)(12)(13)(14) demonstrated that oil washing or sterilization effectively enhances oil quality, and that high-grade oil can remove toxic material from the embryo. Therefore, use of the most appropriate oil appears to be associated with development of high quality embryos.…”

Expression of p-53 and Cox-2 Genes as Tumour Markers in Spontaneous Canine Mammary Tumours

“…The p53 gene mutations and protein overexpression are useful predictors of increased malignant potential and poor prognosis in canine mammary tumours (Lee et al, 2004). The p53 is a significant prognostic marker for survival in early breast cancer in human (Linjawi et al, 2004).…”