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.

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.

SignificanceIatrogenic failures of assisted reproduction technology could be associated with routine sperm preparation techniques. Limitations of conventional sperm selection methods include the inability to efficiently sort functional spermatozoa and assess sperm fertilization potential. We developed a robust microfluidic sperm sorting system by using a diffuser-type microfluidic sperm sorter device capable of ultrahigh-throughput selection and separation of motile, DNA-intact, and functionally competent sperm. The strategy inclusively targeted the intrinsic traits related to fertility and successfully produced livebirths from low-dose insemination of microfluidic sorted spermatozoa. The fertile subpopulation was identified based on the kinetic and trajectory patterns as the sinuous, transitional cohort. The clinical significance of microfluidic sperm sorting is reflected by the established pregnancy and live births of calves.

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 ...