Oxygen Consumption Rate Of Embryos Research Articles

Investigating mitochondria-immune responses in zebrafish, Danio rerio (Hamilton, 1822): A case study with the herbicide dinoseb

The dinitrophenol herbicide dinoseb is an uncoupler of mitochondrial oxidative phosphorylation (OXPHOS). Studies in fish demonstrate impaired OXPHOS is associated with altered immune system responses and locomotor activity in fish. The objective of this study was to determine the effect of dinoseb on zebrafish (Danio rerio) during early stages of development. We measured oxygen consumption rates of embryos, transcripts related to OXPHOS, growth, and the immune system (cytokines and immune-signaling transcripts), and locomotor activity. We hypothesized that OXPHOS of fish would be impaired in vivo, leading to altered basal immune system expression and locomotor activity. Oxidative respiration assessments in embryos revealed that dinoseb decreased both mean basal respiration and oligomycin-induced ATP-linked respiration. Expression levels of cytochrome c oxidase complex IV, 3-hydroxyacyl-COA dehydrogenase and superoxide dismutase 1 were decreased in larvae following exposure to dinoseb while succinate dehydrogenase complex flavoprotein subunit A, insulin growth factor 1 (igf1) and igf2a mRNA were increased in abundance. Immune-related transcripts chemokine (C-X-C motif) ligand 1 and matrix metallopeptidase 9 (MMP-9) were decreased in expression levels while toll-like receptor 5a and 5b were increased in expression. In addition, a visual motor response test was conducted on both 6 and 7 dpf larvae to determine if dinoseb impaired locomotor activity. Dinoseb decreased locomotor activity in 7 dpf larvae but not 6 dpf. This study improves knowledge of toxicity mechanisms for dinoseb in early stages of fish development and demonstrates that mitochondrial toxicants may disrupt immune signaling in zebrafish.

Behavioral and developmental toxicity assessment of the strobilurin fungicide fenamidone in zebrafish embryos/larvae (Danio rerio)

Strobilurin fungicides are among the most widely used in the world and have characteristics that include high water solubility and toxicity to aquatic organisms. While several studies report on mechanisms of toxicity of strobilurins in fish, there are no data on the sub-lethal toxicity of fish to the fungicide fenamidone. To address this gap, survival and hatch rate, deformities, mitochondrial bioenergetics, expression of oxidative stress and apoptotic genes, and behavior (locomotor activity and anxiolytic-related behaviors) were assessed in zebrafish embryos and larvae following exposure to fenamidone. Fenamidone negatively affected development of zebrafish embryos, causing a delay of hatching time at concentrations of 2.5 and 5 μM. Fenamidone caused morphological deformities in zebrafish, including pericardial edema, yolk sac edema, tail deformities, and spinal curvature. Exposure to 1.5 μM fenamidone reduced surface area of swim bladder in larvae at 6 dpf. Fenamidone significantly reduced oxygen consumption rates of embryos; 5 μM fenamidone decreased basal respiration (~85%), oligomycin induced ATP-linked respiration (~70%), FCCP-induced maximal respiration (~75%) and non-mitochondrial respiration (~90%) compared to controls. Sod2 mRNA levels were decreased by fenamidone in larval fish. Locomotor activity was significantly decreased in zebrafish larvae following exposure to 2 μM fenamidone but there was no evidence for anxiolytic nor anxiety-related behaviors (exposures of 100 nM up to 1.5 µM). This study addresses a data gap for potential risks associated with fenamidone exposure in developing fish.

Visual system development and changes in hatching performance in hybrid grouper embryos under different light conditions

Light greatly impacts the performance of teleost embryos. In this study, the development of visual system and performance of Epinehelus moara × Epinephelus lanceolatus embryos under different light spectra (red, orange, green, blue and full spectrum) and dark conditions were assessed. The development of visual system was not completed during embryonic development, characterized by the absence of structural integrity in the retina, indicating the absence of visual photosensitivity. However, nonvisual system may play an important role in photosensitivity. Light conditions contributed significantly to embryo performance, including hatching rate, deformity rate and Wnt signaling pathway expression. There was no statistical difference in the oxygen consumption rate of embryos in different groups during heartbeat onset stage. Embryos in dark group had a significantly lower hatching rate than other groups. The hatching rate in full light spectrum group was significantly higher than red spectrum group, and no significant differences were observed among the other groups. The deformity rate of newly hatched larvae exposed to orange light was significantly lower than those under full spectrum and dark conditions. The mRNA expression level of frizzled2, dickkopf2, limb bud and heart homolog, and natural kermes dye inhibitor of Wnt signaling pathway 1 were detected. The results suggested that dark conditions could lead to the abnormal expression of the Wnt signaling pathway, characterized by significantly lower mRNA expression levels of these factors, which in turn may cause decreased hatchability. In order to increase the hatchability and performance of embryos, and reduce the deformity rate, orange light should be utilized and dark conditions should be avoided during the embryonic development of E. moara × E. lanceolatus.

Developmental neurotoxicity of maneb: Notochord defects, mitochondrial dysfunction and hypoactivity in zebrafish (Danio rerio) embryos and larvae

Broad applications and exposure to the fungicide maneb can lead to toxicity in non-target organisms. Maneb is also associated with neurogenerative diseases such as Parkinson's disease (PD). The objectives of this study were to determine the acute toxicity of maneb to zebrafish by measuring mitochondrial bioenergetics, locomotor activity, and the expression of genes related to the oxidative damage response, as well as those related to dopamine signaling due to its association with PD. Zebrafish embryos at 6 h post-fertilization (hpf) were exposed to either solvent control (0.1% DMSO, v/v), or one dose of 0.1, 0.5, 1.0 and 10.0 µM maneb for 96 h. Maneb was moderately toxic to zebrafish embryos, and had a 96-h LC50 value of 4.29 μM (~ 1.14 mg/L). Maneb induced a dose-dependent increase in mortality, decreased hatching rate, and increased notochord deformity rate at both 1.0 and 10.0 µM after 72 and 96 h. Total body length was also significantly reduced with 1.0 µM maneb. A 50–60% decrease in mean basal oxygen consumption rate was also observed in embryos following a 24 hpf exposure to 10.0 µM maneb but oligomycin-induced ATP production and FCCP-induced maximum respiration remained unaffected. No change was detected in the expression levels of genes associated with oxidative stress (sod1 and sod2), nor those related to dopamine synthesis (th1), dopamine transporter (dat), dopamine receptors (drd1, drd2a, drd3, and drd4b). Thus, modifying the expression of these transcripts may not be a mechanism for maneb-induced developmental toxicity in zebrafish. To assess the potential for neurotoxicity, a dark photokinesis assay was conducted in larvae following 7 d exposure to 0.1, 0.5 and 1.0 μM maneb. Larvae exposed to 0.5 and 1.0 μM maneb showed signs related to hypoactivity, and this reduced activity is hypothesized to be associated with notochord defects as this deformity was prevalent at higher concentrations of maneb. Overall, these data demonstrate that maneb negatively affects embryonic development (i.e. notochord development), affects basal oxygen consumption rates of embryos, and induces hypoactivity in larval fish. This study improves understanding regarding the developmental neurotoxicity of the fungicide maneb to zebrafish.

Standard metabolic rates of early life stages of the diamondback terrapin (Malaclemys terrapin), an estuarine turtle, suggest correlates between life history changes and the metabolic economy of hatchlings

I estimated standard metabolic rates (SMR) using measurements of oxygen consumption rates of embryos and unfed, resting hatchlings of the diamondback terrapin (Malaclemys terrapin) three times during embryonic development and twice during the early post-hatching period. The highest observed SMRs occurred during mid to late embryonic development and the early post-hatching period when hatchlings were still reliant on yolk reserves provided by the mother. Hatchlings that were reliant on yolk displayed per capita SMR 135 % higher than when measured 25 calendar days later after they became reliant on exogenous resources. The magnitude of the difference in hatchling SMR between yolk-reliant and exogenously feeding stages was much greater than that attributed to costs of digestion (specific dynamic action) observed in another emydid turtle, suggesting that processing of the yolk was not solely responsible for the observed difference. The pre-feeding period of yolk reliance of hatchlings corresponds with the period of dispersal from the nesting site, suggesting that elevated SMR during this period could facilitate dispersal activities. Thus, I hypothesize that the reduction in SMR after the development of feeding behaviors may reflect an energy optimization strategy in which a high metabolic expenditure in support of development and growth of the embryo and dispersal of the hatchling is followed by a substantial reduction in metabolic expenditure coincident with the individual becoming reliant on exogenous resources following yolk depletion.

Development of a new clinically applicable device for embryo evaluation which measures embryo oxygen consumption

Does a new system-the chip-sensing embryo respiration monitoring system (CERMs)-enable evaluation of embryo viability for potential application in a clinical IVF setting? The system enabled the oxygen consumption rate of spheroids, bovine embryos and frozen-thawed human embryos to be measured, and this rate corresponded to the developmental potential of embryos. To date, no reliable and clinically suitable objective evaluation methods for embryos are available, which circumvent the differences in inter-observer subjective view. Existing systems such as the scanning electrochemical microscopy (SECM) technique, which enables the measurement of oxygen consumption rate in embryos, need improvement in usability before they can be applied to a clinical setting. This is a prospective original research study. The feasibility of measuring the oxygen consumption rate was assessed using CERMs for 9 spheroids, 9 bovine embryos and 30 redundant frozen-thawed human embryos. The endpoints for the study were whether CERMs could detect a dissolved oxygen gradient with high sensitivity, had comparable accuracy to the SECM measuring system with improved usability, and could predict the development of an embryo to a blastocyst by measuring the oxygen consumption rate. The relationship between the oxygen consumption rate and standard morphological evaluation was also examined. We developed a new CERMs, which enables the oxygen consumption rate to be measured automatically using an electrochemical method. The device was initially used for measuring a dissolved oxygen concentration gradient in order to calculate oxygen consumption rate using nine spheroids. Next, we evaluated data correlation between the CERMs and the SECM measuring systems using nine bovine embryos. Finally, the oxygen consumption rates of 30 human embryos, which were frozen-thawed on 2nd day after fertilization, were measured by CERMs at 6, 24, 48, 72 and 96 h after thawing with standard morphological evaluation. Furthermore, the developed blastocysts were scored using the blastocyst quality score (BQS), and the correlation with oxygen consumption rate was also assessed. The device enabled the oxygen consumption rate of an embryo to be measured automatically within a minute. The oxygen concentration gradient profile showed excellent linearity in a distance-dependent change. A close correlation in the oxygen consumption rates of bovine embryos was observed between the SECM measuring system and CERMs, with a determination coefficient of 0.8203 (P =0.0008). Oxygen consumption rates of human embryos that have reached the blastocyst stage were significantly higher than those of arrested embryos at 48, 72 and 96 h after thawing (P =0.039, 0.004 and 0.049, respectively). Thus, in vitro development of frozen-thawed human embryos to the blastocyst stage would be predicted at 48 hafter thawing (day 4) by measuring the oxygen consumption using CERMs. Although a positive linear relationship between BQS and the oxygen consumption rate was observed [the determination coefficient was R(2)=0.6537 (P =0.008)], two blastocysts exhibited low oxygen consumption rates considering their relatively high BQS. This suggests that morphology and metabolism in human embryos might not correlate consistently. Transfer of the embryo and pregnancy evaluation was not performed. Thus, a correlation between oxygen consumption and the in vivo viability of embryos remains unknown. Clinical trials, including embryo transfer, would be desirable to determine a threshold value to elect clinically relevant, quality embryos for transfer. We utilized frozen-thawed human embryos in this study. The effect of these manipulations on the respiratory activity of the embryo is also unknown. Selection of quality embryos, especially in a single embryo transfer cycle, by CERMs may have an impact on obtaining better clinical outcomes, albeit with clinical trials being required. Furthermore, the early determination of quality embryos by CERMs may enable the omission of long-term in vitro embryo culture to the blastocyst stage. CERMs is scalable technology that can be integrated into incubators and/or other embryo evaluation systems, such as the time-lapse systems, due to its chip-based architecture. Thus, CERMS would enable automatic measurement of oxygen consumption, under 5% CO2, in the near future, in order to reduce oxidative stress from exposure to atmospheric air. This study was supported by grants from the Health and Labor Sciences Research Grant (H24-Hisaichiiki-Shitei-016). The authors have no conflicts of interest. Not applicable.

The relationship between oxygen consumption rate and viability of in vivo-derived pig embryos vitrified by the micro volume air cooling method

The aim of this study was to assess the viability of vitrified-warmed in vivo-derived pig embryos after measuring the oxygen consumption rate. Six days after artificial insemination, blastocysts were collected from gilts and vitrified by the micro volume air cooling method. The oxygen consumption rate was measured in 60 vitrified-warmed embryos, which were then cultured for 48h to assess the viability. The survival (re-expansion) rate of embryos after warming was 85.0%. The average oxygen consumption rate of embryos immediately after warming was greater in embryos which could re-expand during subsequent culture (F=0.75±0.04) than that in those which failed to re-expand (F=0.33±0.05). Moreover, the oxygen consumption rate of vitrified-warmed embryos was greater in the hatched (F=0.88±0.06) than that in the not-hatched group (F=0.53±0.04). When the oxygen consumption rate of the vitrified-warmed embryos and the numbers of viable and dead cells in embryos were determined, there was a positive correlation between the oxygen consumption rate and the number of live cells (P<0.01, r=0.538). A total of 29 vitrified embryos after warming and measuring the oxygen consumption rate were surgically transferred into uterine horns of two recipients. Both of the recipients become pregnant and farrowed 12 healthy piglets. These results demonstrate that the oxygen consumption rate of vitrified-warmed pig embryos can be related to the number of live cells and that the measurement of oxygen consumption of embryos after cryopreservation may be useful for estimating embryo survivability.

Relationships between oxygen consumption rate, viability, and subsequent development of in vivo–derived porcine embryos

Oxygen consumption rate of in vivo–derived porcine embryos was measured, and its value as an objective method for the assessment of embryo quality was evaluated. Embryos were surgically collected 5 or 6 days after artificial insemination (AI), and oxygen consumption rate of embryos was measured using an embryo respirometer. The average oxygen consumption rate (F × 1014/mol s−1) of the embryos that developed to the compacted morula stage on Day 5 (Day 0 = the day of artificial insemination) was 0.58 ± 0.03 (mean ± standard error of the mean). The Day-6 embryos had consumption rates of 0.56 ± 0.13, 0.87 ± 0.06, and 1.13 ± 0.07 at the early blastocyst, blastocyst, and expanded blastocyst stages, respectively, showing a gradual increase as the embryos developed. Just after collection, the average oxygen consumption rates of embryos that hatched and of those that did not hatch after culture were 0.60 ± 0.04 and 0.50 ± 0.04 for Day 5 (P = 0.08) and 1.05 ± 0.09 and 0.77 ± 0.05 for Day 6 (P < 0.05), respectively. The value and probability of discrimination by measuring the oxygen consumption rates of embryos to predict their hatching ability after culture were 0.56 and 63.6% for Day-5 embryos and 0.91 and 68.4% for Day-6 blastocysts, respectively. When Day-5 embryos were classified based on the oxygen consumption rate and then transferred non-surgically to recipient sows, three of the seven sows, to which embryos having a high oxygen consumption rate (≥0.59) were transferred, became pregnant and farrowed a total of 20 piglets. However, none of the four sows, to which embryos having low oxygen consumption rate (<0.59) were transferred, became pregnant. These results suggest that the viability of in vivo–derived porcine embryos and subsequent development can be estimated by measuring the oxygen consumption rate.

Novel automated device with the chip electrode for monitoring respiratory activity of embryos

Morphological evaluation has been generally used to evaluate embryo quality. However, it is subjective and frequently different between investigators. We previously reported that the oxygen consumption rate of embryos measured by the scanning electrochemical microscopy was a useful parameter for evaluating embryo quality1). Although this method is an epoch for high sensitivity and non-invasiveness, popularization of the device is stagnant because it takes a lot of time to handle the device. We have developed a new device that measures the oxygen consumption rate automatically.

138 FATTY ACID β-OXIDATION REGULATES BOVINE EMBRYO OXYGEN METABOLISM

Mammalian oocytes are rich in endogenous lipid, which provides a potential source of metabolic energy during oocyte maturation and early embryo development after fertilisation. Despite this, studies on early embryo metabolism have focussed on consumption of substrates from the culture medium, with comparatively little consideration for endogenous stores. Additionally, the interaction between fatty acid β-oxidation and oxygen consumption during pre-implantation development has not been investigated. This study has investigated the relationship between embryo fatty acid metabolism and oxygen consumption rate (OCR), a marker of overall oxidative metabolic activity. The amount of oxygen consumption by bovine blastocysts coupled to ATP synthesis was assessed by measuring embryo OCR following acute treatment with oligomycin, which inhibits ATP synthase. Bovine embryos were then produced in vitro and cultured from zygote to blastocyst stage (8 days) in the presence of β-mercaptoacetate (BMA), a competitive inhibitor of fatty acid β-oxidation, or L-carnitine, a cofactor that promotes β-oxidation. OCR of individual embryos was measured using a noninvasive, highly sensitive oxygen probe (Unisense), whereas OCR of embryos in groups was determined using the Oxygen Biosensor (OBS) fluorimetric assay (BD Biosciences, Erembodegem, Belgium). Mean OCR measured by each technique correlated well. Data are presented as means ± SEM. The effect of acute inhibition were compared by a paired t-test, with pre-treated embryos acting as their own control, whereas the effect of L-carnitine and BMA on OCR was compared across treatment groups using ANOVA with Dunn’s test for unequal groups post hoc, using Sigmaplot (Systat Software Inc., San Jose, CA, USA). Treatment with oligomycin caused blastocyst OCR to fall from 23.5 ± 3.3 to 7.9 ± 2.5 pmol/embryo/h (n = 3; P = 0.03). Inhibition of β-oxidation by BMA led to a modest, but consistent increased OCR in cleavage stage embryos: 4-cell: 14.8 ± 7.2 (n = 12) v. 8.9 ± 1.1 pmol/embryo/h (n = 3) control; 8-cell: 15.8 ± 2.2 (n = 6) v. 11.9 ± 2.3 pmol/embryo/h (n = 3) control. At the morula stage, BMA led to a significant rise in OCR (26.8 ± 3.6 (n = 7) v. 7.8 ± 1.7 pmol/embryo/h (n = 3) control; P = 0.003) and at the blastocyst stage, OCR was significantly elevated in the presence of BMA; 41.3 ± 2.6 (n = 19) v. 16.9 ± 1.3 pmol/embryo/h (n = 14) control (P = 0.01). By contrast, when groups of embryos were cultured in the presence of L-carnitine, blastocyst OCR was reduced by 45% from 24.7 ± 5.1 (group control) to 15.9 ± 3.7 pmol/embryo/h (n = 30; P &lt; 0.06). We report that 66.5% of oxygen consumption by bovine blastocysts is oligomycin sensitive, meaning that 33.5% of oxygen consumption is used for processes other than ATP synthesis. Surprisingly, inhibition of fatty acid β-oxidation caused total bovine embryo OCR to rise, although the reasons for this are unclear. It is possible that inhibiting β-oxidation leads to an accumulation of free fatty acids in the mitochondrion, which can disrupt the inner membrane and lead decoupling of ATP synthesis from oxygen consumption. Thus, manipulating fatty acid oxidation in the early embryo dysregulates oxidative metabolism; the implications of this are under further investigation.

Measurement of oxygen consumption rate of embryos to select the best embryo for e-SET

OBJECTIVE: Respiration is a useful parameter for evaluating embryo quality as it provides important information about metabolic activity. The scanning electrochemical microscopy (SECM) measuring system provides a non-invasive, simple, accurate, and consistent measurement of the respiration activity of single human embryos. In this study, we examined the clinical efficacy for IVF-elected single embryo transfer (eSET) patients using the SECM measuring system. DESIGN: prospective randomised study. MATERIALS AND METHODS: A total of 68 IVF-eSET patients were treated under the prospective randomized schedule at our private clinic. After morphological evaluation by the Veeck's method, oxygen consumption by individual human embryos was quantified by SECM measuring system. A single embryo, based on conventional morphological evaluation or morphological evaluation plus measuring the oxygen consumption rate, was elected by either decision and transferred. RESULTS: Among the cycles (N=72) that embryos showed morphologically exactly same grade by Veeck's method, the pregnancy rate in cycles with an embryo elected based on morphological evaluation plus measuring the oxygen consumption rate had a significantly higher pregnancy rate of 50.0%(18/36). compare with an embryo elected by conventional morphological method was 25.0%(9/36). Further more, the abortion rate of the oxygen measured group(5.6% 1/18) was less than the conventional group(22.2% 2/9). Finally, on-going pregnancy rate was significantly higher in the oxygen measured group(47.2% 17/36) than the conventional morphological method group(19.4% 7/36). CONCLUSION: The results of this study, indicated the high pregnancy rate and low abortion rate will come to fruition by electing the best embryo by morphological evaluation plus measuring the oxygen consumption rate in IVF-eSET. It is hoped that this new, novel method will prove to be more effective for IVF treatment.

Viability of Porcine Embryos after Vitrification Using Water-soluble Pullulan Films

The efficiency of a porcine embryo vitrification method that uses water-soluble films of pullulan, a naturally-occurring polysaccharide polymer, was compared with two other types of vitrification methods using different devices and solutions for vitrification and warming. Blastocysts collected in vivo and vitrified by the conventional straw (ST), Cryotop((R)) (MVC) or pullulan film vitrification (PFV) methods were stored in liquid nitrogen for a certain period of time, after which the cryoprotective agents were removed by stepwise dilution. Fresh embryos were used as controls for the non-vitrification group. The vitrified-warmed embryos were incubated in TCM199 with 0.1 mM beta-mercaptoethanol and 20% fetal bovine serum for 24 h at 38.5 C in humidified air with 5% CO(2) to evaluate their viability. The survival rate of embryos in the ST group (48.3%) was significantly lower than that of those in the MVC (70.7%), PFV (79.0%) and non-vitrification (94.4%) groups. The oxygen consumption rate after vitrification was significantly lower than that before vitrification in the ST group, but was not significantly different in the MVC and PFV groups. Both the oxygen consumption rates of embryos after warming and the live cell numbers in the ST group were lower than those in the MVC group, while they did not differ significantly between the PFV and MVC groups. There was a correlation between the oxygen consumption rate and the number of live cells in vitrified embryos after warming. Our results demonstrated that in vivo-derived porcine embryos could be vitrified using pullulan films.

Quantifying oxygen diffusion in paraffin oil used in oocyte and embryo culture

Oxygen diffusion through oil is important in the culture of oocytes and embryos. A diffusion coefficient two orders of magnitude smaller than that of oxygen in water has been thought possible, and this has led to concerns of anoxia in cultures. Using an assay for determining the oxygen consumption rate of embryos and oocytes, along with a mathematical model, it is here shown that the oxygen diffusion rate in paraffin oil at 37 degrees C is about two-thirds of that in water at the same temperature. Although not previously recognised for the assay in question, the geometry is such that anoxia does occur for a period of time in excess of 1 hr and, by the completion of the assay, 30-40% of the medium is anoxic. Hence the quantity of oxygen consumed is less than would be consumed in conditions of plentiful oxygen supply. Nevertheless, using a model with a concentration dependent oxygen consumption rate, the oxygen consumption rate can be estimated.

Microclimate of nesting burrows of the Rainbow Bee-eater

Burrow-nesting affords protection from predators and climatic extremes, but potentially can pose physiological ‘problems’ for developing birds and attendant adults. Microclimate parameters of burrows of breeding Rainbow Bee-eaters (Merops ornatus) were measured to assess whether they presented such difficulties for young and adults. Estimated mean volume of the brood-chamber was ∼4.5 L. Relative humidity was typically 100% in the brood-chamber and chamber air temperature was constant, averaging 4–6°C above ambient levels. The temperature regime of the burrow probably resulted in low thermoregulatory costs for attendant adults and endothermic nestlings. The chamber oxygen (O2) fraction (mean 19.35%) was always lower than ambient values, but mostly not sufficiently low to be problematic for the growing young. Mean pre-internal pipping absolute oxygen consumption rate of embryos (62.9 ± 13.8 mL O2 day−1) did not appear to be strongly influenced by either the protracted incubation period or the reduced O2 partial pressure of the chamber atmosphere. Mean eggshell water-vapour conductance (8.95 mg day−1 kPa−1) was tuned to egg mass rather than egg mass/incubation period. However, eggs still lost ∼15% of their mass during incubation because the influence of the small water-vapour pressure difference across the shell (2.91 kPa) and the protraction of the incubation period apparently counteracted each other.

The effect of some water parameters on the oxygen consumption rate of embryos and larvae of the Chinese Sturgeon (Acipenser sinensis)

The effect of some water parameters on the oxygen consumption rate of embryos and larvae of the Chinese Sturgeon (Acipenser sinensis)

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

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&amp;apos;s PBS containing 1.5 M ethylene glycol, 0.1 M trehalose, and 20&amp;percnt; calf serum. They were thawed by holding the straws in air for 8 s and then immersing them in a 30&amp;deg;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&amp;percnt; fetal bovine serum and 0.1 mM &amp;beta;-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 &amp;lt; 0.05) than those of CM collected on the same day (0.84 vs. 1.29 &amp;times; 10&amp;minus;14 mol s&amp;minus;1, respectively). A significant difference was also observed in consumption rates between grade 1 and 2 embryos at the BL stage (P &amp;lt; 0.05). After freezing&amp;ndash;thawing, the average oxygen consumption rates of embryos were 0.52 &amp;times; 10&amp;minus;14 mol s&amp;minus;1 for CM (n &amp;equals; 9), 0.67 &amp;times; 10&amp;minus;14 mol s&amp;minus;1 for EB (n &amp;equals; 8), and 0.96 &amp;times; 10&amp;minus;14 mol s&amp;minus;1 for BL (n &amp;equals; 11). The CM embryos with rates of &amp;lt; 0.5 &amp;times; 10&amp;minus;14 mol s&amp;minus;1 and the EB and BL embryos with those &amp;lt; 0.6 &amp;times; 10&amp;minus;14 mol s&amp;minus;1 did not show good morphological appearance after 24 h in culture. Pregnant animals were not obtained from embryos with rates &amp;lt;0.5 &amp;times; 10&amp;minus;14 mol s&amp;minus;1 for CM (n &amp;equals; 5) and &amp;lt;0.7 &amp;times; 10&amp;minus;14 mol s&amp;minus;1 for EB (n &amp;equals; 9). A high pregnancy rate (67&amp;percnt;) was obtained from embryos with rates &amp;gt;1.0 &amp;times; 10&amp;minus;14 mol s&amp;minus;1 for BL (n &amp;equals; 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.

Relationship between Oxygen Consumption and Sex of Bovine In Vitro Fertilized Embryos

The present study was conducted to examine the relationship between the oxygen consumption rate and sex ratio of bovine in vitro fertilized embryos on each day of blastocyst formation. The quality of blastocysts collected on day 7, 8, and 9 after in vitro fertilization (IVF) were categorized as ranks A and B (excellent and good, respectively) based on microscopic observation of the morphology. The oxygen consumption rate and sex of individual blastocysts were evaluated using two novel techniques: scanning electrochemical microscopy (SECM) and loop-mediated isothermal amplification (LAMP), respectively. The oxygen consumption rates of embryos of rank A were significantly higher (p < 0.05) than those of rank B, irrespective of the day of blastocyst appearance after IVF. Neither did the proportion of male embryos of ranks A and B differ significantly from each other at any of the days examined, nor from the average proportion (53%). The oxygen consumption rate of embryos of rank B collected on day 8 was significantly higher (p < 0.05) in female embryos than in male embryos collected on the same day. However, there were no apparent differences of oxygen consumption rates at each day of blastocyst appearance between male and female embryos of rank A. These results indicate that the oxygen consumption rate of individual embryos reflects their quality but does not correlate with the sex ratio of embryos of excellent quality.

Oxygen consumption rates of grunion ( Leuresthes tenuis) embryos exposed to the petroleum hydrocarbon, benzo[ a]pyrene

Bioconcentration of [ 14C]benzo[ a]pyrene and effects of unlabeled benzo[ a]pyrene (BaP) accumulation on the routine oxygen consumption of embryonic grunion ( Leuresthes tenuis) were studied. At Day 15, bioconcentration factors over dissolved BaP levels ranged from 249 to 466. Weight-specific respiration rates at Days 14–15 were significantly increased ( P ≦ 0.05) at a mean BaP body burden of 0.51 ppm wet wt. Oxygen consumption rates of embryos containing 0.70 to 12.80 ppm BaP were not significantly different from control rates. Because in a previous study embryos containing 0.51 ppm BaP exhibited hatching and developmental rates similar to those of controls, their metabolic response to low-level hydrocarbon exposure may be an example of hormesis, an overcompensating metabolic regulation to inhibitory challenges.

Oxygen consumption of a large, cold-adapted frog egg (Ascaphus truei (Amphibia: Ascaphidae))

Female Ascaphus lay a small number of large eggs beneath rocks in cold, montane streams of the Pacific Northwest. Average number of eggs per female is 45 (range, 28 to 64) and average egg diameter is 4 mm (range, 3.5 to 4.5). The large eggs of Ascaphus contain a relatively large amount of nutrients. Average wet weight of a single egg is 37.5 mg and dry weight is 23.7 mg (63% of wet weight). The oxygen consumption rate of embryos during development at 15 °C increases gradually during cleavage (0.25 μl O2/h per embryo) and more rapidly during neurula and postneurula stages (0.60 to 0.90 μl O2/h per embryo) to a value of 1.20 μl O2/h per embryo at vitelline circulation. The cold-adapted eggs of Ascaphus are characterized by large size, slow developmental rate and low oxygen consumption rate.