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...

We have developed a polystyrene-based well-of-the-well (WOW) system using injection molding to track individual embryos throughout culture using time-lapse cinematography (TLC). WOW culture of bovine embryos following in vitro fertilization was compared with conventional droplet culture (control). No differences between control- and WOW-cultured embryos were observed during development to the blastocyst stage. Morphological quality and inner cell mass (ICM) and trophectoderm (TE) cell numbers were not different between control- and WOW-derived blastocysts; however, apoptosis in both the ICM and TE cells was reduced in WOW culture (P < 0.01). Oxygen consumption in WOW-derived blastocysts was closer to physiological level than that of control-derived blastocysts. Moreover, WOW culture improved embryo viability, as indicated by increased pregnancy rates at Days 30 and 60 after embryo transfer (P < 0.05). TLC monitoring was performed to evaluate the cleavage pattern and the duration of the first cell cycle of embryos from oocytes collected by ovum pickup; correlations with success of pregnancy were determined. Logistic regression analysis indicated that the cleavage pattern correlated with success of pregnancy (P < 0.05), but cell cycle length did not. Higher pregnancy rates (66.7%) were observed for animals in which transferred blastocysts had undergone normal cleavage, identified by the presence of two blastomeres of the same size without fragmentation, than among those with abnormal cleavage (33.3%). These results suggest that our microwell culture system is a powerful tool for producing and selecting healthy embryos and for identifying viability biomarkers.

Abstract. The aim of this study was to develop an in-straw dilution method suitable for 1-step bovine embryo transfer of vitrified embryos using the Cryotop vitrification-straw dilution (CVSD) method. The development of embryos vitrified using the CVSD method was compared with those of embryos cryopreserved using in-straw vitrificationdilution (ISVD) and conventional slow freezing, outside dilution of straw (SFODS) methods. In Experiment 1, in vitroproduced (IVP) embryos cryopreserved using the CVSD method were diluted, warmed and exposed to the dilution solution at various times. When vitrified IVP embryos were exposed to the dilution solution for 30 min after warming, the rates of embryos developing to the hatched blastocyst stage after 72 h of culture (62.0-72.5%) were significantly lower (P<0.05) than those of embryos exposed to the solution for 5 and 10 min (82.4-94.3%), irrespective of supplementation with 0.3 M sucrose in the dilution solution. In Experiment 2, the rate of embryos developing to the hatching blastocyst stage after 48 h of culture in IVP embryos cryopreserved using the SFODS method (75.0%) was significantly (P<0.05) lower than those of embryos cryopreserved using the CVSD and ISVD methods (93.2 and 97.3%, respectively). In Experiment 3, when in vivo-produced embryos that had been cryopreserved using the CVSD, ISVD and SFODS methods and fresh embryos were transferred to recipient animals, no significant differences were observed in the conception and delivery rates among groups. In Experiment 4, when IVP embryos derived from oocytes collected by ovum pick-up that had been cryopreserved using the CVSD and ISVD methods and fresh embryos were transferred to recipient animals, no significant differences were observed in the conception rates among groups. Our results indicate that this simplified regimen of warming and diluting Cryotop-vitrified embryos may enable 1-step bovine embryo transfer without the requirement of a microscope or other laboratory equipment. Key words: Bovine, Conception rate, Cryotop, Embryo transfer, Vitrification (J. Reprod. Dev. 57: [437][438][439][440][441][442][443] 2011) ince the first successful cryopreservation of bovine embryos [1], cryopreservation of bovine embryos has been widely used commercially. A recent worldwide inventory revealed that more than 250,000 bovine in vivo-produced embryos have been used for embryo transfer (ET) after freezing and thawing [2]. However, the pregnancy rate of frozen-thawed embryos is slightly lower than that of fresh embryos [3]. And the pregnancy rate of frozen-thawed in vitro-produced (IVP) embryos is also significantly lower than that of fresh IVP embryos. Therefore, it is necessary to develop an embryo cryopreservation method to obtain higher conception rates. Recent reports have confirmed that vitrification of embryos, especially IVP embryos, is at least as efficient as conventional slow freezing [4][5][6][7]. Vitrification reduces the time commitment and equipment expense associated with cryopreservation compared with con...

Abstract. Mitochondrial bioenergetics in mammalian oocytes has not been sufficiently characterized. In this study, the function of oxidative phosphorylation (OXPHOS), a major pathway in mitochondria, was investigated in individual bovine oocytes by monitoring oxygen consumption using modified scanning electrochemical microscopy (SECM). At the germinal vesicle (GV) stage, 65% of basal respiration was used for mitochondrial respiration, which was inhibited by complex IV inhibitor. Around 63% of mitochondrial respiration was coupled to ATP synthesis, as determined by sensitivity to an ATP synthase inhibitor, and the remaining 37% was attributed to proton leak. In contrast, 50% and 43% of mitochondrial respiration were used for ATP synthesis in in vivo-and in vitro-derived metaphase II (MII)-stage oocytes, respectively. ATP-linked respiration, in both in vivo-and in vitro-derived MII-stage oocytes, was significantly lower than in GV-stage oocytes, suggesting that OXPHOS in bovine oocytes is more active at the GV stage compared with the MII stage. Interestingly, basal respiration in in vitro-derived MII oocytes was significantly higher than for in vivo-derived oocytes, reflecting an increase in proton leak. Next, we assessed respiration in MII oocytes cultured for 8 h. The aged oocytes had a significantly reduced maximum respiratory capacity, which was stimulated by a mitochondrial uncoupler, and reduced ATP-linked respiration compared with non-aged oocytes. However, the aging-related phenomenon could be prevented by caffeine treatment. We conclude that OXPHOS in bovine oocytes varies in the transition from GV to MII stage, in vitro maturation and the aging process. This approach will be particularly useful for analyzing mitochondrial bioenergetics in individual mammalian oocytes. Key words: Bovine, Mitochondrial function, Oocyte, Oxidative phosphorylation, Oxygen consumption (J. Reprod. Dev. 58: [636][637][638][639][640][641] 2012) M itochondria play fundamental roles in the cell, and mitochondrial dysfunction has been linked with several pathologies, including infertility and developmental failure. Although they share general characteristics, mitochondria can have distinct features based on inner membrane invaginations and matrix structures. Depending on their cell type and functional status, mitochondria present an extensive range of morphologies, are functionally heterogeneous [1], and vary in number [2]. Oxidative phosphorylation (OXPHOS), the process that couples substrate oxidation to ATP synthesis, is the major and best-known metabolic function of mitochondria. During OXPHOS, electrons are transferred from nutrients to reducing equivalents (e.g., NADH), then to electron carriers, and finally to oxygen. Such electron transfer is mediated by oxido-reductive reactions of the tricarboxylic acid cycle in the mitochondrial matrix and by electron transport in the inner mitochondrial membrane. The energy harvested during these oxido-reductive reactions is stored in a proton gradient across the inner mitochondrial ...