Establishment of An Advanced Chemically Defined Medium for Early Embryos Derived from In Vitro Matured and Fertilized Bovine Oocytes

Abstract

Abstract.A chemically defined medium would be useful for analyzing promoters or inhibitors in in vitro culture (IVC) of bovine embryos. However, an IVC system for bovine embryos in a chemically defined medium has not been fully established. The present study was carried out to establish an advanced chemically defined medium for bovine embryos that supports a high rate of embryo development to the blastocyst stage. In the first experiment, we examined the effects of addition of Medium RD (RPMI1640 and Dulbecco's MEM, 1:1 v/v) to mKSOM/aa on developmental competence. The addition of 10% RD to mKSOM/aa with BSA improved the rate of development to the blastocyst stage; however, 10% RD-mKSOM/aa with PVP, which is a chemically defined medium, caused a reduction in the percentage of hatching blastocysts. In the second experiment, embryos were cultured in the chemically defined medium of 10% RD-mKSOM/ aa containing 11.7, 23.4, 46.8, 70.2 or 96.8 μM inositol. Inositol at the concentration of 70.2 μM improved the rate of development to the hatching blastocyst stage. In the third experiment, the optimal RD concentration in the IVC medium was evaluated. Embryos were cultured in the chemically defined medium supplemented with 10, 20 or 30% (v/v) RD. The rate of development to the blastocyst stage was highest with 20% RD. In the fourth experiment, the effects of Nacetylglucosamine (GlcNAc) as an IVC medium supplement on developmental competence were examined. The rate of development to the blastocyst stage with 1.0 mM GlcNAc was significantly higher than that without GlcNAc, but the rate of development with 1.2 mM GlcNAc was not different from that without GlcNAc. We also evaluated the ability of blastocysts produced in RD-mKSOM/aa to develop to normal calves after being transferred into recipients. Ten of the 16 recipients became pregnant, with 9 delivering normal calves. These results indicate that 20% RD-mKSOM/aa containing 70.2 μM myo-inositol and 1 mM GlcNAc is useful as a chemically defined medium for IVC of bovine embryos.Key words: Bovine embryos, Chemically defined medium, GlcNAc, Myo-inositol (J. Reprod. Dev. 57 : [681][682][683][684][685][686][687][688][689] 2011) I n vitro production (IVP) of bovine embryos by an in vitro maturation-fertilization culture system has been greatly improved. However, the rate of development of bovine oocytes to the blastocyst stage following maturation, fertilization and culture in vitro is limited to about 30-40% [1]. One possible reason for this is that embryo culture conditions, particularly the culture medium used for embryos, are suboptimal. Optimal conditions of a culture medium for embryos are 1) a high rate of embryo development to the blastocyst stage with repeatable results and 2) production of highquality blastocysts that have the competence to develop into competence to normal calves. In most studies on culture of mammalian embryos, the culture medium is supplemented with serum. A beneficial effect of serum on the development of bovine oocytes to the blastocyst stage ha...

Enzymatic activity of mouse group X-sPLA2 improves in vitro production of preimplantation bovine embryos

“…In this study, we therefore investigated the impact of adding recombinant mGX, a catalytically inactive mutant of mGX and the bovine group IB sPLA2 during in vitro oocyte maturation, fertilization and early preimplantation development of bovine embryos, on initial cleavage, production of blastocyst and blastocyst hatching rates. dibekacin sulfate 65 µg/mL [29], and covered with 250 µL equilibrated mineral oil. Embryos were maintained in culture for 9 days.…”

Usefulness of modified Medium RD as a chemically defined medium for in vitro maturation of bovine oocytes

“…In vitro fertilization (IVF) was performed according to Momozawa and Fukuda. 10 Briefly, one straw (0.5 mL/straw) of frozen semen of Japanese Black cattle was thawed in a water bath at 39°C for 20 seconds, suspended in 5 mL of fertilization medium with 10 mmol/L caffeine (sodium benzoate, Sigma-Aldrich), and washed twice by centrifugation at 700 g for 5 min. The fertilization medium was modified BO (HEPES-BO, bovine serum albumin-free).…”

“…The embryo culture medium used as a chemically defined medium in the present study was 20% RD-mKSOM/aa. 10 Between 120 and 144 hours after insemination, embryos at the morula stage or a more advanced stage were transferred to a fresh culture medium supplemented with 50 μmol/L β-ME. The morphological quality of embryos at blastocyst stage was evaluated following the criteria of IETS manual.…”

Change in energy metabolism of in vitro produced embryos: an alternative to make them more cryoresistant?

“…A culture medium modification using defined or semi-desiccated media without serum did not affect the blastocyst yield and increased their survival post cryopreservation (MUCCI et al, 2006;BLOCK et al, 2010;MOMOZAWA;FUKUDA, 2011). According to Sudano et al (2011), only a reduction in the FCS concentration in the culture medium is sufficient to decrease the lipid content and increase post-cryopreservation embryo survival.…”