22 RESPIRATORY ACTIVITY AND ULTRASTRUCTURAL FEATURES OF BOVINE SOMATIC NUCLEAR TRANSFER EMBRYOS

2007 ◽  
Vol 19 (1) ◽  
pp. 129
Author(s):  
H. Abe ◽  
K. Aoyagi ◽  
S. Aoyagi ◽  
H. Shiku ◽  
T. Matsue ◽  
...  
Keyword(s):  
Oxygen Consumption ◽  
Nuclear Transfer ◽  
Respiratory Activity ◽  
Measuring System ◽  
Ionophore A23187 ◽  
Bovine Embryos ◽  
Single Donor ◽  
Close Relationship ◽  
Consumption Rates ◽  
Functional Peptides

We succeeded in determining oxygen consumption of individual bovine embryos non-invasively and quantitatively by scanning electrochemical microscopy (SECM). Recently, we have found that there is a close relationship between high oxygen consumption and developmental ability of bovine IVF embryos. However, the relationship between the respiratory activity and the quality of bovine embryos reconstructed by somatic cell nuclear transfer (SCNT) is still unclear. The aims of this study were: (1) to assess the oxygen consumption of single bovine SCNT and IVF embryos; and (2) to examine the ultrastructural features of SCNT embryos. Bovine oocytes were matured in IVMD101 medium (Research Institute for the Functional Peptides, Yamagata, Japan) and enucleated. The recipient oocytes were activated by treatment with Ca ionophore A23187 and then incubated with IVMD101 containing cycloheximide. Single donor cells (fibroblasts derived from an adult cow) were placed into the perivitelline space of the enucleated oocytes and the two cells were fused by electrofusion. The nuclear transfer embryos were cultured in IVD101 medium without bovine cumulus/granulosa cell co-culture in a humidified atomosphere of 5% CO2/95% air at 38.5�C. Oxygen consumption rates by whole embryos were quantified individually by the SECM measuring system. Some of these embryos were prepared for observation by transmission electron microscopy. Statistical analyses were performed using one-way analysis of variance (ANOVA) and Fisher

204 RELATIONSHIP BETWEEN RESPIRATORY ACTIVITY AND THE PREGNANCY RATE OF BISECTED BOVINE EMBRYOS IN VIVO

2007 ◽  
Vol 19 (1) ◽  
pp. 219 ◽  
Author(s):  
S. Moriyasu ◽  
H. Hirayama ◽  
K. Sawai ◽  
S. Kageyama ◽  
S. Aoyagi ◽  
...  
Keyword(s):  
Oxygen Consumption ◽  
Pregnancy Rate ◽  
Embryo Transfer ◽  
Respiratory Activity ◽  
Pregnancy Rates ◽  
Bovine Embryos ◽  
Chi Square ◽  
Important Indicator ◽  
Consumption Rates

Oxygen consumption is an important indicator of the metabolic activity of living cells, which may provide valuable information for evaluating embryo quality. We have found that the bovine embryos with high oxygen consumption possess stronger potential for further development. However, the relationship between respiratory activity and the pregnancy rate of embryos is still unclear. In this study, we investigated the respiration rates of bisected bovine embryos and the pregnancy rates of demi-embryos after embryo transfer. Compact morula-stage embryos were bisected evenly by micro glass needle. One hundred bisected embryos were incubated for 24 h in embryo culture medium (IVD101; Research Institute for the Functional Peptides, Yamagata, Japan) at 39�C under 5% CO2, 5% O2, 90% N2. After the incubation, demi-embryos were classified into 2 groups: blastocoel-formed (BC) and blastocoel-not-formed (CM) embryos. Oxygen consumption rates of demi-embryos were measured by scanning electrochemical microscopy (SECM; Hokuto Denko Corporation, Tokyo, Japan). Within 3 h after the measurement, 80 demi-embryos were transferred into recipient cows (one demi-embryo/one recipient) at 7–8 days after estrus. Recipient cows were diagnosed for pregnancy by ultrasonography approximately 40 days after estrus. Statistical difference was analyzed by Tukey's post-hoc test and chi-square test. A total of 27 recipient cows became pregnant; the pregnancy rates for cows with CM and BC demi-embryos were 40.6% (13/32) and 29.2% (14/48), respectively. Mean oxygen consumption rates (� 10-14 mol s-1) in pregnant and non-pregnant cows were 0.47 and 0.39 for CM demi-embryos and 0.63 and 0.52 for BC demi-embryos, respectively. Retrospective analysis showed that the respiratory activity of demi-embryos in the pregnant group was higher than those in the non-pregnant group. In particular, the pregnancy rates for demi-embryos with respiratory activity higher than 0.35 in CM and 0.40 in BC groups were 52.0% (13/25) and 35.9% (14/39), respectively. On the other hand, cows with demi-embryos having an oxygen consumption rate under 0.35 in CM (n = 7) and 0.40 in BC (n = 9) groups did not become pregnant. These results demonstrated that bovine demi-embryos with higher respiratory activity showed a high pregnancy rate after embryo transfer. It is generally known that the pregnancy rate after the transfer of bisected embryos is lower than that of whole embryos. The measurement of oxygen consumption by SECM procedures is a useful tool to assess the quality of pre-implantation embryos and may contribute to the improvement of the success rate for bisected embryo transfer.

scholarly journals 129 RESPIRATION ACTIVITY OF BOVINE EMBRYOS CULTURED IN SERUM-FREE AND SERUM-CONTAINING MEDIA

2005 ◽  
Vol 17 (2) ◽  
pp. 215 ◽  
Author(s):  
H. Abe ◽  
H. Shiku ◽  
S. Aoyagi ◽  
T. Matsue ◽  
H. Hoshi
Keyword(s):  
Oxygen Consumption ◽  
Oxygen Consumption Rate ◽  
Embryo Quality ◽  
Consumption Rate ◽  
Developmental Stages ◽  
Culture Conditions ◽  
Bovine Embryos ◽  
Respiration Activity ◽  
Serum Free ◽  
Consumption Rates

Oxygen consumption is a ubiquitous parameter which can provide valuable information about metabolic mechanisms and embryo quality. Recently, we succeeded in non-invasively and quantitatively determining oxygen consumption of individual bovine embryos by the scanning electrochemical microscopy (SECM). The aim of this study was to assess by SECM the oxygen consumption of individual bovine embryos at different developmental stages cultured in serum-free and serum-supplemented media. Bovine oocytes were matured in IVMD101 medium [Research Institute for the Functional Peptides (IFP), Shimojo, Yamagata, Japan] and inseminated in BO-based medium. For serum-free culture, inseminated ooocytes were cultured to the blastocyst stage in IVD101 medium in an atmosphere of a low oxygen condition (5% CO2/5% O2/90% N2) at 38.5°C. For serum-supplemented culture, inseminated oocytes were cultured in HPM199 medium (IFP) supplemented with 5% calf serum (HPM199 + CS) in the presence of bovine cumulus/granulosa cells in a humidified atmosphere of 5% CO2 in air. Oxygen consumption by individual bovine embryos was non-invasively quantified by the SECM measuring system. Some embryos were prepared for transmission electron microscopy. The oxygen consumption rates are presented in the table. Oxygen consumption rates (F) of the single embryos were low from 2-cell to 8-cell stages (0.45–0.52 × 10−14 mol s−1). In serum-free culture, an increase in oxygen consumption rate was found at the morula (1.03 × 10−14 mol s−1) stage, and blastocysts showed an even higher oxygen consumption rate (1.86 × 10−14 mol s−1). On the other hand, the oxygen consumption of morulae and blastocysts produced in serum-supplemented medium was lower than that of embryos cultured in serum-free medium. Electron microscopic study demonstrated that many of the mitochondria of morulae and blastocycts cultured in HPM199 + CS medium were an immature form, indicating a correlation between respiration activity and development of mitochondria. These results suggest that the culture conditions affect the respiration activity of bovine embryos. The SECM procedures may have a wide application for judging embryo quality and culture conditions for embryos. Table 1. Oxygen consumption rates (F × 10−14 mol s−1) of the bovine embryos at various developmental stages

216 THE RELATIONSHIP BETWEEN OXYGEN CONSUMPTION RATE AND PREGNANCY RATE OF BOVINE EMBRYOS

2007 ◽  
Vol 19 (1) ◽  
pp. 225 ◽  
Author(s):  
N. Sakagami ◽  
K. Akiyama ◽  
Y. Nakazawa
Keyword(s):  
Oxygen Consumption ◽  
Pregnancy Rate ◽  
Embryo Transfer ◽  
Embryo Quality ◽  
Calf Serum ◽  
Pregnancy Rates ◽  
Bovine Embryos ◽  
Significant Difference ◽  
Consumption Rates ◽  
The Relationship

A precise evaluation of embryo quality is important to estimate the suitability of embryo transfer to recipient animal. Recently, an objective evaluation method was reported for bovine embryos, in which the oxygen consumption of embryos can be noninvasively determined by scanning electrochemical microscopy (SECM) (Shiku et al. 2001 Anal. Chem. 73, 3751–3758). Trimarchi et al. (2000 Biol. Reprod. 62, 1866–1874) suggested that the oxygen consumption reflects the cell number and mitochondrial activity of embryos. The objectives of this study were (1) to examine the oxygen consumption of in vivo-derived embryos by SECM, (2) to investigate the relationship between oxygen consumption and morphological estimation of embryos, and (3) to assess the correlation among the oxygen consumption, embryo viability, and pregnancy rates. Fifty-six embryos were collected from Japanese Black cattle, which were superovulated with a total dose of 20 mg porcine FSH (FSH-R; Kawasaki Pharmaceutical Co., Ltd., Tokyo, Japan) followed by AI. The qualities of collected embryos at the stage of compacted morulae (CM), early blastocysts (EB), and blastocysts (BL) on Day 7 after AI were categorized as grade 1 and grade 2, according to the IETS manual (2002). The oxygen consumption rates of embryos were evaluated by SECM, as previously described by Abe et al. (2004 J. Mamm. Ova Res. 21). Embryos were frozen by programmable freezer in Dulbecco's PBS containing 1.5 M ethylene glycol, 0.1 M trehalose, and 20% calf serum. They were thawed by holding the straws in air for 8 s and then immersing them in a 30°C water bath for 15 s. After thawing, the embryos were examined for oxygen consumption. Twenty-eight embryos were then cultured in TCM-199 supplemented with 20% fetal bovine serum and 0.1 mM β-mercaptoethanol for 24 h to assess the viability of embryos by re-expansion of blastocole. The remaining 28 embryos were transferred to recipients. The pregnancy rates were determined by rectal palpation on Day 70. Data were analyzed by ANOVA. The consumption rates of BL embryos on Day 7 were significantly higher (P < 0.05) than those of CM collected on the same day (0.84 vs. 1.29 × 10−14 mol s−1, respectively). A significant difference was also observed in consumption rates between grade 1 and 2 embryos at the BL stage (P < 0.05). After freezing–thawing, the average oxygen consumption rates of embryos were 0.52 × 10−14 mol s−1 for CM (n = 9), 0.67 × 10−14 mol s−1 for EB (n = 8), and 0.96 × 10−14 mol s−1 for BL (n = 11). The CM embryos with rates of < 0.5 × 10−14 mol s−1 and the EB and BL embryos with those < 0.6 × 10−14 mol s−1 did not show good morphological appearance after 24 h in culture. Pregnant animals were not obtained from embryos with rates <0.5 × 10−14 mol s−1 for CM (n = 5) and <0.7 × 10−14 mol s−1 for EB (n = 9). A high pregnancy rate (67%) was obtained from embryos with rates >1.0 × 10−14 mol s−1 for BL (n = 14). These results suggest that the measurement of oxygen consumption of embryos after embryo freezing and prior to embryo transfer may be useful for estimating embryo quality and suitability of embryo transfer.

319 OXYGEN CONSUMPTION OF BOVINE CUMULUS CELLS AND OOCYTES CULTURED IN DIFFERENT CULTURE SYSTEMS FOR OOCYTE MATURATION

2006 ◽  
Vol 18 (2) ◽  
pp. 267 ◽  
Author(s):  
H. Abe ◽  
H. Shiku ◽  
S. Aoyagi ◽  
T. Matsue ◽  
H. Hoshi
Keyword(s):  
Oxygen Consumption ◽  
Oocyte Maturation ◽  
Calf Serum ◽  
Cumulus Cells ◽  
Culture Conditions ◽  
Measuring System ◽  
Electron Microscopic ◽  
Respiration Activity ◽  
Serum Free ◽  
Consumption Rates

Oxygen consumption is a ubiquitous parameter that can provide valuable information on metabolic mechanisms and on oocyte and embryo quality. Recently, we succeeded in non-invasively and quantitatively determining oxygen consumption of individual bovine embryos by scanning electrochemical microscopy (SECM). The aim of this study was to assess by SECM the oxygen consumption of bovine cumulus cells and oocytes cultured in serum-free and serum-supplemented media for oocyte maturation. Bovine cumulus–oocyte complexes (COCs) were obtained from ovarian follicles 2–6 mm in diameter. COCs were cultured in IVMD101 medium for serum-free culture and HPM199 medium supplemented with 5% calf serum (HPM199+CS) for serum-supplemented culture in a humidified atmosphere of 5% CO2 in air (20% O2) at 38.5°C for 24 h. Oxygen consumption by single COCs was non-invasively quantified by a SECM measuring system (Abe et al. 2004 J. Mamm. Ova Res. 21, 22). After the measurements, COCs were treated with 0.5% pronase to completely remove the cumulus cells. The oxygen consumption of single denuded oocyte was measured by SECM. Some COCs and oocytes were prepared for transmission electron microscopy. Oxygen consumption has been monitored in COCs and oocytes cultured in IVMD101 and HPM199+CS media for oocyte maturation (Table 1). Oxygen consumption rates of the immature COCs and denuded oocytes (immediately upon recovery from ovary: control) were 6.91 and 0.70 (×10−14 mol s−1), respectively. In serum-free culture (IVMD101), an increase in oxygen consumption rate was found in oocytes, whereas the oxygen consumption of COCs decreased during oocyte maturation. On the other hand, the oxygen consumption of COCs and oocytes cultured in serum-supplemented medium (HPM199+CS) were not change compared with that of controls. Electron microscopic study demonstrated that the mitochondria moved from a peripheral location in the ooplasm to an even spatial distribution in the oocytes cultured in IVMD101 medium, whereas many of the mitochondria in oocytes cultured in HPM199+CS were distributed in the peripheral region of the ooplasm after oocyte maturation. These results suggest that the respiration activity of bovine cumulus cells and oocytes changed during oocyte maturation, and the respiration activity and ultrastructural features of oocytes may affect the culture conditions. The SECM procedures may provide valuable information on oocyte quality and culture conditions for oocyte maturation. Table 1. Oxygen consumption rates (F × 10−14 mol s−1) of the bovine COCs and oocytes in oocyte maturation cultures

A new photometric method for oxygen consumption measurements in cell suspensions

1986 ◽  
Vol 61 (2) ◽  
pp. 449-455 ◽  
Author(s):  
W. Mueller-Klieser ◽  
R. Zander ◽  
P. Vaupel
Keyword(s):  
Volume Fraction ◽  
High Sensitivity ◽  
Cell Suspensions ◽  
Measuring System ◽  
Photometric Method ◽  
O2 Consumption ◽  
Physical Density ◽  
O2 Concentration ◽  
Consumption Rates ◽  
Time Required

A new technique is described for measuring O2 consumption rates and O2 concentrations in suspensions of respiring cells. Aliquots of a cell suspension kept in a special thermostated precision syringe are injected into the measuring system in defined time intervals. The O2 content of these samples is determined photometrically, as reported previously. The O2 consumption per cellular wet weight and/or per single cell can be calculated from the cell volume fraction, the physical density, the cell concentration in the suspension, and the time-dependent decline of the O2 concentration in the precision syringe. The minimum detectable amount of O2 is 0.1 microliter O2, which corresponds to 0.001 (vol/vol) of O2 if a 100-microliters sample of suspended cells is analyzed. Reproducibility of the O2 consumption measurement is 9% of the measured value. The advantages offered by this method are the straightforward calibration in absolute terms, the short time required for one analysis (2–6 min), a high sensitivity, the simultaneous determination of overall O2 concentration and O2 consumption rates in cell suspensions, and the great variability in the application.

Viability and respiratory activity ofPseudomonas syringaecells starved in buffer

1995 ◽  
Vol 41 (4-5) ◽  
pp. 372-377 ◽  
Author(s):  
João P. S. Cabral
Keyword(s):  
Oxygen Consumption ◽  
Electron Transport ◽  
Pseudomonas Syringae ◽  
Respiratory Activity ◽  
Bacterial Cells ◽  
Intact Cells ◽  
Rate Of Oxygen Consumption ◽  
Metabolic Activities ◽  
Different Levels ◽  
Number Of Cells

Pseudomonas syringae cells starved in buffer released orcinol-reactive molecules and materials that absorbed ultraviolet light. The number of cells culturable in nutrient medium decreased more rapidly than the number of intact particles determined by microscopy. The results suggested that starvation resulted in the lysis of an increasing number of cells, and that a fraction of the intact particles were not culturable. Starvation also resulted in a decrease in the rate of oxygen consumption with acetate, glycerol, and succinate, but at different levels. Whereas the respiration of acetate and glycerol decreased concomitantly with culturability, the respiration of succinate decreased to levels similar to the concentration of intact cells, suggesting that all intact particles respired the succinate, but only the culturable cells respired the acetate and glycerol. The results suggest that measuring the activity of the electron-transport system can overestimate the viability of starved bacterial cells, and that complex metabolic activities such as the respiration of acetate and glycerol are probably better suited for the evaluation of this parameter.Key words: Pseudomonas syringae, starvation, culturability, viability, respiration.

scholarly journals Oxygen consumption rates in subseafloor basaltic crust derived from a reaction transport model

2013 ◽  
Vol 4 (1) ◽  
Author(s):  
Beth N. Orcutt ◽  
C. Geoffrey Wheat ◽  
Olivier Rouxel ◽  
Samuel Hulme ◽  
Katrina J. Edwards ◽  
...  
Keyword(s):  
Oxygen Consumption ◽  
Transport Model ◽  
Consumption Rates ◽  
Reaction Transport ◽  
Basaltic Crust

The Relation of Oxygen Consumption to Body Size and to Temperature in the Larvae of Chironomus Riparius Meigen

1958 ◽  
Vol 35 (2) ◽  
pp. 383-395
Author(s):  
R. W. EDWARDS
Keyword(s):  
Oxygen Consumption ◽  
Body Size ◽  
Dry Matter ◽  
Larval Instar ◽  
Weight Ratio ◽  
Dry Weight ◽  
Chironomus Riparius ◽  
Consumption Rates ◽  
Rate Of Oxygen Consumption ◽  
Wet Weight

1. The oxygen consumption rates of 3rd- and 4th-instar larvae of Chironomus riparius have been measured at 10 and 20° C. using a constant-volume respirometer. 2. The oxygen consumption is approximately proportional to the 0.7 power of the dry weight: it is not proportional to the estimated surface area. 3. This relationship between oxygen consumption and dry weight is the same at 10 and at 20° C.. 4. The rate of oxygen consumption at 20° C. is greater than at 10° C. by a factor of 2.6. 5. During growth the percentage of dry matter of 4th-instar larvae increases from 10 to 16 and the specific gravity from 1.030 to 1.043. 6. The change in the dry weight/wet weight ratio during the 4 larval instar supports the theory of heterauxesis. 7. At 20° C., ‘summer’ larvae respire faster than ‘winter’ larvae.

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