Conventionally, in vitro–fertilized (IVF) bovine embryos are morphologically evaluated at the time of embryo transfer to select those that are likely to establish a pregnancy. This method is, however, subjective and results in unreliable selection. Here we describe a novel selection system for IVF bovine blastocysts for transfer that traces the development of individual embryos with time-lapse cinematography in our developed microwell culture dish and analyzes embryonic metabolism. The system can noninvasively identify prognostic factors that reflect not only blastocyst qualities detected with histological, cytogenetic, and molecular analysis but also viability after transfer. By assessing a combination of identified prognostic factors—(i) timing of the first cleavage; (ii) number of blastomeres at the end of the first cleavage; (iii) presence or absence of multiple fragments at the end of the first cleavage; (iv) number of blastomeres at the onset of lag-phase, which results in temporary developmental arrest during the fourth or fifth cell cycle; and (v) oxygen consumption at the blastocyst stage—pregnancy success could be accurately predicted (78.9%). The conventional method or individual prognostic factors could not accurately predict pregnancy. No newborn calves showed neonatal overgrowth or death. Our results demonstrate that these five predictors and our system could provide objective and reliable selection of healthy IVF bovine embryos.

Abstract. This study was conducted to study the kinetics of initial cell divisions in relation with the cleavage patterns in viable (with the ability to develop to the blastocyst stage) and non-viable bovine embryos and parthenotes. The kinetics of in vitro development and cleavage patterns were observed by time lapse cinematography. The length of the first and second but not third cell cycle differed significantly between the viable and non-viable embryos after IVF or parthenogenesis. Viable embryos had significantly shorter first and second cell cycles than non-viable ones. The presence of fragments, protrusions and unequally-sized blastomeres was associated with an extended one-cell stage and reduced ability to develop to the blastocyst stage; however, the lengths of the second and third cell cycles were not altered. Oocytes showing direct division from one cell to 3 or 4 blastomeres showed similar developmental ability and embryonic cell numbers to those showing normal division, although, with a high frequency of chromosomal abnormalities. Our results suggest that the differences in the first cell cycles between viable and non-viable embryos were not sperm-related, whereas direct cleavage of 1-cell embryos to 3 or more blastomeres and protrusion formation are related to sperm-driven factors. The length of the first and second cell cycles and the cleavage pattern should be examined simultaneously to predict developmental competence of embryos at early cleavage stages. Key words: Bovine, Cleavage pattern, Developmental kinetics, Embryo, In vitro culture, Time lapse cinematography (J. Reprod. Dev. 56: [200][201][202][203][204][205][206][207] 2010) ecent progress in the improvement of in vitro embryo production (IVP) systems allows us to produce large quantities of bovine embryos [1]. Nevertheless, the developmental competence of embryos produced by IVP is still inferior to that of in vivo produced embryos due to by different stresses resulting from the imperfection of the IVP systems in imitating in vivo conditions [2,3]. Selection of embryos with good developmental competence is essential to achieve high success rates by transfer of embryos, especially when only one or a very few embryos are transferred into a surrogate mother at one time, such as in humans and cattle. The transfer of mammalian embryos at early cleavage stages (e.g., at the 1-8 cell stage) may have the advantage of avoiding stresses caused by the culture systems. Human embryos produced by assisted reproduction technology are still often selected and transferred at the cleavage stage [4]. Finding the most reliable marker of developmental competence in early embryos is still a matter of quest and debate among researchers. The number, morphology and position of pronuclei are often evaluated to estimate embryo quality in humans [5]; however, controversial data regarding these selection markers have also been reported [6,7]. Moreover oocytes of large farm animals such as pigs and cattle contain a high number of lipid droplets that completely blo...

High lipid content in embryos is associated with low freezing tolerance. This study assessed the effects of exogenous L-carnitine, an enhancer of lipid metabolism, on the in vitro development and freezing survival of bovine embryos. Also, effects on metabolic activity, reactive oxygen species (ROS) and apoptosis were investigated. Supplementation of embryo culture medium with 1.518 mM or 3.030 mM L-carnitine significantly increased the rates of zygote development to the blastocyst stage and blastocyst cell numbers whereas 6.072 mM of this compound did not improve embryo development. Survival rates after slow freezing of blastocysts were significantly higher when embryos were cultured in the presence of 1.518 mM or 3.030 mM L-carnitine compared with the control. A lower density of lipid droplets was detected in L-carnitine-treated blastocysts compared with the control. L-carnitine significantly reduced ROS levels in 2-cell embryos but did not reduce ROS levels at later stages. The apoptotic cell rate was not different between control and L-carnitine-treated blastocysts. L-carnitine significantly increased ATP levels in 2-cell embryos but not at the 8-cell or blastocyst stages. L-carnitine increased the expression of metabolism-related ATP6 and COX1 genes in blastocysts. In conclusion, L-carnitine supplementation enhanced lipid metabolism in embryos resulting in improved development and cryotolerance of bovine blastocysts produced in vitro.