scholarly journals Respiration rates of individual bovine in vitro-produced embryos measured with a novel, non-invasive and highly sensitive microsensor system

Reproduction ◽  
2005 ◽  
Vol 130 (5) ◽  
pp. 669-679 ◽  
Author(s):  
A S Lopes ◽  
L H Larsen ◽  
N Ramsing ◽  
P Løvendahl ◽  
M Räty ◽  
...  
Keyword(s):  
Respiration Rate ◽  
Embryo Quality ◽  
Developmental Stages ◽  
Embryo Morphology ◽  
The Novel ◽  
Non Invasive ◽  
Respiration Rates ◽  
Morphological Evaluation ◽  
Highly Sensitive

Oxygen consumption is a useful parameter for evaluating embryo quality, since it provides a valuable indication of overall metabolic activity. Over the years, several approaches have been used to measure the respiration rates of individual embryos, but a convincing method has not yet been reported. In this study, we introduce and have validated a novel high resolution microsensor technology to determine the respiration rates of individual embryos at different developmental stages. We have employed this technology to investigate the correlation between respiration rate and embryo morphology, diameter and sex. Following morphological evaluation, individual respiration rates of day 3 (n = 18) and day 7 (n = 60) bovine in vitro-produced embryos were determined. Of the measured embryos, 64 were lysed for sex diagnosis by PCR. Average respiration rates of day 7 embryos (1.30 ± 0.064 nl/h) were 3.4-fold higher than day 3 embryos (0.38 ± 0.011 nl/h). On day 7, the average respiration rate of quality 1 blastocysts was significantly higher than the respiration rates of the lower qualities. For both day 3 and day 7 embryos, respiration rates were directly influenced by embryo diameter but did not differ between sexes. These results have demonstrated that the novel microsensor technology can be used to accurately and rapidly (8 min) measure the respiration rates of individual embryos at different developmental stages. Respiration rates were only in partial agreement with embryo morphology, suggesting a slight discrepancy between these two methods in assessing embryo quality. It is likely that a combined assessment of embryo respiration and morphology would improve embryo classification and subsequent selection.

scholarly journals Analysis of Morphokinetic Parameters of Feline Embryos Using a Time-Lapse System

Animals ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 748
Author(s):  
Joanna Kochan ◽  
Agnieszka Nowak ◽  
Barbara Kij ◽  
Sylwia Prochowska ◽  
Wojciech Niżański
Keyword(s):  
Embryo Development ◽  
Embryo Quality ◽  
Time Lapse ◽  
Blastocyst Stage ◽  
Cavity Formation ◽  
Non Invasive ◽  
Morphokinetic Parameters ◽  
Morphological Defects ◽  
Development In Vitro

The aim of this study was to analyze the morphokinetic parameters of feline embryos using a time lapse system. Oocytes matured in vitro were fertilized (IVF) and in vitro cultured in a time lapse-system (Primo Vision®, Gothenburg, Sweden). The first cell division of embryos occurred between 17 h post insemination (hpi) and 38 hpi, with the highest proportion of embryos (46%) cleaving between 21 and 24 hpi. The timing of the first cleavage significantly affected further embryo development, with the highest development occurring in embryos that cleaved at 21–22 hpi. Embryos that cleaved very early (17–18 hpi) developed poorly to the blastocyst stage (2%) and none of the embryos that cleaved later than 27 hpi were able to reach the blastocyst stage. Morphological defects were observed in 48% of the embryos. There were no statistically significant differences between the timing intervals of the first cleavage division and the frequency of morphological defects in embryos. Multiple (MUL) morphological defects were detected in more than half (56%) of the abnormal embryos. The most frequent single morphological defects were cytoplasmic fragmentation (FR) (8%) and blastomere asymmetry (AS) (6%). Direct cleavage (DC) from 1–3 or 3–5 blastomeres, reverse cleavage (RC) and vacuoles were rarely observed (2–3%). The timing of blastocyst cavity formation is a very good indicator of embryo quality. In our study, blastocyst cavity formation occurred between 127–167 hpi, with the highest frequency of hatching observed in blastocysts that cavitated between 142–150 hpi. Blastocysts in which cavitation began after 161 h did not hatch. In conclusion, the timing of the first and second cleavage divisions, the timing of blastocyst cavity formation and morphological anomalies can all be used as early and non-invasive indicators of cat embryo development in vitro.

scholarly journals 2 CORRELATION BETWEEN OXYGEN RESPIRATION RATES AND MORPHOLOGY, SEX, DIAMETER AND DEVELOPMENTAL STAGE OF SINGLE BOVINE IVP-EMBRYOS

2005 ◽  
Vol 17 (2) ◽  
pp. 151 ◽  
Author(s):  
A.S. Lopes ◽  
N. Ramsing ◽  
L.H. Larsen ◽  
M. Räty ◽  
J. Peippo ◽  
...  
Keyword(s):  
Developmental Stage ◽  
Respiration Rate ◽  
Developmental Stages ◽  
Glass Tube ◽  
Outer Diameter ◽  
Bovine Embryos ◽  
Embryo Viability ◽  
Concentration Gradients ◽  
Cell Stage ◽  
Respiration Rates

A simple, non-invasive, rapid and sensitive oxygen microsensor system was developed to investigate correlations between oxygen respiration rates of individual bovine embryos and their morphology, sex, diameter and developmental stage. Bovine IVP-embryos (n = 78; Holm et al. Theriogenology 52, 683–700) were analysed around the 8-cell stage (Day 3; n = 18) and at various blastocyst stages (Day 7; n = 60). Each embryo was morphologically evaluated, its outer diameter measured and was then loaded into a glass tube (i.d. 0.68 mm, length 3 mm). After 1 h, oxygen concentration gradients generated by the embryo’s respiration were measured over app. 8 min with an oxygen microelectrode (www.unisense.com). Five embryos were measured in one round together with an empty tube as control. The procedure was repeated twice for each embryo with app. 1 h interval. Individual respiration rates in nL O2/embryo/h (nL/h) were calculated from these gradients. The measurements were performed at 38.5°C under constant flow of humidified 5% CO2 in air (app. 19% O2). After this, 64 embryos (14 Day 3; 50 Day 7) were lysed for sex diagnosis by PCR. Values are given as mean ± SD. The sensitivity of the oxygen measurement system was high (controls: 0.034 ± 0.035 nL/h, n = 15) and its repeatability from 1st to 2nd measurement was 99.7 ± 9.8% (n = 71). The average embryo respiration rate was 0.39 ± 0.05 nLl/h on Day 3 (n = 18) and 1.31 ± 0.52 nLl/h on Day 7 (n = 60). For Day 7 embryos, the respiration rates varied according to their morphological quality, being 1.87 ± 0.46a (n = 18), 1.17 ± 0.32b (n = 23), 0.95 ± 0.27b,c (n = 14) and 0.72 ± 0.24c (n = 4) nL/h for quality 1, 2, 3, and 4 embryos, respectively (Proc Mixed,a,b,c: P < 0.05; values with different superscripts differ significantly). The sex ratio (male:female) was 9:5 (Day 3) and 32:18 (Day 7), and on Day 7 this ratio varied between qualities: 11:2, 12:8, 8:4, and 1:3 for quality 1, 2, 3, and 4, respectively. The average respiration rate on day 3 was the same for males and females, as it was on day 7 (1.22 ± 0.43 nL/h (females) and 1.31 ± 0.58 nL/h (males), P > 0.05). There was a correlation between embryo diameter and respiration rate (r2 = 0.65, n = 74), which was even stronger for Day 7 male embryos (r2 = 0.72, n = 32). In conclusion, a highly reliable, repeatable and sensitive system was established for measuring respiration rates in single bovine embryos, even at early developmental stages. The respiration rate was lower on day 3 compared to Day 7 embryos, and it was correlated with the morphological embryo quality on Day 7. Oxygen consumption could be a valuable supplementary indicator of embryo viability, especially in difficult evaluations (e.g. quality 2 and 3 after IVP). It remains to be demonstrated if such measurements can also reveal quality differences already at Day 3, which would be of interest in, e.g. the human field. ASL is supported by FCT, Portugal.

Evaluating Headline Fungicide on Alfalfa Production and Sensitivity of Pathogens to Pyraclostrobin

2013 ◽  
Vol 14 (1) ◽  
pp. 10 ◽  
Author(s):  
Deborah A. Samac ◽  
Bill Halfman ◽  
Bryan Jensen ◽  
Fritz Brietenbach ◽  
Lisa Behnken ◽  
...  
Keyword(s):  
Return On Investment ◽  
Crude Protein ◽  
Field Experiments ◽  
Developmental Stages ◽  
Forage Yield ◽  
Fungicide Application ◽  
Highly Sensitive ◽  
Disease Pressure ◽  
Foliar Pathogens

Headline fungicide was recently registered for management of foliar diseases on alfalfa. The effect of disease control on yield, forage quality, and potential return on investment for fungicide application was determined for field experiments conducted at five locations in 2012, three in Wisconsin and two in Minnesota. Headline reduced defoliation in 12 out of 14 harvests and increased forage yield and return on investment in 5 out 12 harvests compared to the untreated control. Headline plus the insecticide Warrior II reduced defoliation in 10 out of 14 harvests and increased yield and return on investment in four harvests compared to Warrior II alone. Two common foliar pathogens were highly sensitive to pyraclostrobin, the active ingredient in Headline. For Phoma medicaginis, the in vitro EC50 was 2.3 ng pyraclostrobin/ml and for Stemphylium globuliferum the EC50 was 52 ng pyraclostrobin/ml. The results indicate that fungicide application can increase yields under higher disease pressure and increase crude protein when the crop is harvested at later developmental stages. Accepted for publication 25 June 2013. Published 17 September 2013.

scholarly journals Effect of resveratrol on the in vitro maturation of ovine (Ovis aries) oocytes and the subsequent development of handmade cloned embryos

2019 ◽  
Vol 5 (4) ◽  
Author(s):  
José Luis Martínez-Ibarra ◽  
Eugenia Adriana Espinoza-Mendoza ◽  
Raymundo Rangel-Santos ◽  
Demetrio Alonso Ambriz-García ◽  
María Del Carmen Navarro-Maldonado
Keyword(s):  
Embryo Quality ◽  
Developmental Stages ◽  
Subsequent Development ◽  
Poor Quality ◽  
Ovis Aries ◽  
Control Group ◽  
Nuclear Maturation ◽  
Significant Difference ◽  
Maturation Rate

The effect of resveratrol on the in vitro maturation (IVM) of ovine (Ovis aries) oocytes and the development of handmade cloned embryos was evaluated. The nuclear maturation and reactive oxygen species (ROS) levels in the oocytes, as well as the early development and morphological cloned embryo quality, were evaluated under different resveratrol concentrations (0, 0.5, 2 and 5 μM). After IVM, no significant difference was observed in the maturation rate of oocytes treated with 0.5 μM (81.3 %) and 2 μM (72 %) resveratrol compared to that of the control group (0 μM) (74.2 %), but the rate significantly decreased at 5 μM (56 %) (p < 0.05). When the oocyte ROS levels were determined, no significant differences among the groups were observed (p > 0.05). For cloned embryo development, the embryos obtained from the oocytes treated with 0.5 μM resveratrol showed higher (p < 0.05) compacted morula rates (10.7 %) compared to the embryos obtained from the oocytes treated with 0, 2 and 5 μM (6.2, 0 and 0 %, respectively). Regarding embryo morphological quality, the embryos from the oocytes treated with 0.5 μM resveratrol showed a lower rate of poor quality morulae (4.7 %) in comparison to those treated with 0, 2 and 5 μM (23.8, 23.3 and 33.3 %, respectively) (p < 0.05). In conclusion, resveratrol showed no significant improvement on the IVM or ROS levels in domestic ovine oocytes. However, treatment with 0.5 μM resveratrol during IVM improved embryo quality and promoted morulae compaction of Ovis aries handmade cloned embryos.Figure 3. Different developmental stages of the HMC sheep embryos cultured in the WOW system. Cleaved embryos (a-d), 8‒16 blastomere embryos (e-h), morulae (i-l) and compact morulae (m-p) (200X).

scholarly journals Non-invasive assessment of in-vitro embryo quality to improve transfer success

2015 ◽  
Vol 31 (5) ◽  
pp. 585-592 ◽  
Author(s):  
Tina Rødgaard ◽  
Peter M.H. Heegaard ◽  
Henrik Callesen
Keyword(s):  
Embryo Quality ◽  
Non Invasive ◽  
Transfer Success

scholarly journals Morphological Evaluation and Grading of Human Embryo Quality from Day - 1 to Day - 3 Embryos for Optimum Conceiving Rate

2020 ◽  
pp. 225-236
Author(s):  
Ram Dayal ◽  
Sumer Singh ◽  
Prabhat Kumar ◽  
Kamla Singh ◽  
Pawan Kumar Tripathi ◽  
...  
Keyword(s):  
Cell Size ◽  
Embryo Transfer ◽  
Embryo Development ◽  
Human Embryo ◽  
Embryo Quality ◽  
Implantation Rate ◽  
Grading System ◽  
Morphological Evaluation ◽  
Early Compaction

<p>A grading system of human embryo is very important for embryo selection & predicting blastocyst formation from day - 1, day 2, & day – 3, were sequentially explained. We were designed a methods for grading of embryos from day – 1 (zygote as a pronuclear evaluation, PN), day – 2 (cleavage stage) and day - 3 (evaluation of developmental stage according to cell size and fragmentation) embryo according to cell size and degree of fragmentation and early compaction. In the first grading system pronuclear study and poly-spermy, Second grading system is based on the blastomere / cell number and the observation of fragmentation pattern and selection for embryo transfer, embryos vitrification and pregnancy outcome. Assessment of embryo quality in order to select the embryos that have higher chance to give pregnancy, it is critical goal in IVF cycle or assisted reproductive technologies. ET current trend in human infertility treatment with IVF / ICSI embryo transfer (IVF / ICSI ET) is to increase the chance of higher pregnancy and reduce the multiple pregnancies after multiple embryo transfer according to patient age and endometrium thickness as well as own ART Laboratory protocol. Morphological evaluation & grading of human embryo as a reliable and no-invasive method that provides valuable information & prediction of IVF/ICSI embryos which has developmental potential to reach till early compaction or blastocyst. This research paper describes the current status of morphological embryo evaluation from zygote to eight cell blastomeres or early compaction on late day 3.</p> <p>We found higher embryo development potential and early compaction during in-vitro embryo culture conditions and higher implantation rate in Grade A embryos in comparison transferred of Grade B embryos. Overall embryo development in-vitro and conceiving rate was seen <strong>48.06 %</strong> after embryo transfer of both grades A and Grade B embryos in 233 patients in different age group with different endometrium thickness and multiple embryos transfer in one uterus depend on patient previous history. <strong>935</strong><strong>Embryos was selected from 2702 developing embryos for embryo transfer (ET)</strong> was performed in <strong>233 patients. </strong>After this study we found implantation rate (IR) was <strong>48.06%</strong> based on embryo quality, morphology and grade.</p>

scholarly journals 155OXYGEN MEASUREMENTS AS INDICATORS OF CULTURE CONDITIONS AND VIABILITY OF CATTLE EMBRYOS

2004 ◽  
Vol 16 (2) ◽  
pp. 199 ◽  
Author(s):  
A.S. Lopes ◽  
L.D.M. Ottosen ◽  
T. Greve ◽  
H. Callesen
Keyword(s):  
Oxygen Consumption ◽  
Oxygen Partial Pressure ◽  
Partial Pressure ◽  
Respiration Rate ◽  
Embryo Morphology ◽  
Atmospheric Conditions ◽  
In Vitro Production ◽  
Embryo Viability ◽  
Routine Handling

Experiments were performed to study oxygen and temperature conditions experienced by embryos during routine in vitro production (IVP) (Exp. 1) and to evaluate oxygen consumption of single embryos as a viability indicator (Exp. 2). In both experiments, bovine IVP embryos were used (Holm et al. 1999, Theriogenology 52: 683–700), and a combined oxygen-temperature microsensor (tip diameter approx. 50μm) was used to attain oxygen partial pressure gradients and temperature profiles. In Exp. 1, 4-well dishes with in vitro culture (IVC) medium (400-μL or 50-μL droplets) covered with 400μL oil were taken from the incubator (5% CO2 , 5% O2 , 90% N) and measured under atmospheric air conditions at room temperature (24–25°C). The profiles were determined over 2min, starting 2min after the dishes were taken out of the incubator. The 400μL medium was measured from the top layer and gradually 3/4 down into the well (seven replicates). The oxygen partial pressure decreased from 21.0±0.66% O2 at the top layer to 15.0±1.6% O2 at the bottom layer, while the average temperature increased from 27.2±3.0°C to 31.7±0.7°C. In the 50-μL droplets, the profiles were obtained in the middle of the droplet (five replicates). The oxygen partial pressure was 17.1±2.44% O2 and the temperature 31.0±1.11°C. Consequently, routine handling of culture dishes outside the incubator seems to have a strong influence on both temperature and oxygen conditions. In Exp. 2, Day 3 and Day 7 embryos were evaluated morphologically before being loaded consecutively into one of the holes in a measuring block developed for this purpose. The block was previously covered with 40mL IVC medium before being incubated for 3 days under 5% CO2 , 95% N (38.5°C, 100% humidity). The oxygen consumption of each embryo was measured from 75±5min after removal from the incubator. Each measurement lasted approx. 5min, was repeated three times for each embryo, and was performed under atmospheric conditions on a warming stage held at 37±1°C. The average oxygen consumption by Day 3 embryos was 0.25±0.14nL/embryo/h (n=20), and no clear relation between respiration rate and embryo morphology could be demonstrated. For Day 7 embryos, the average oxygen consumption was 0.90±0.56nL/embryo/h (n=22), and this varied according to their morphological quality (1.17±0.70 (n=7), 0.98±0.49 (n=6) and 0.46±0.38 (n=9) nL/embryo/h for good, fair and poor quality embryos, respectively). In conclusion, this study illustrated the changes in O2 partial pressure that embryos can temporarily be exposed to under routine handling, even for rapid procedures such as cleavage inspection. The results also show that respiration rate is lower in Day 3 than in Day 7 embryos. Furthermore, oxygen consumption values of Day 7 embryos seem to be in agreement with the morphological embryo quality, supporting the idea that oxygen consumption can be a valuable parameter for the evaluation of embryo viability.

Non-invasive mitochondrial DNA quantification on Day 3 predicts blastocyst development: a prospective, blinded, multi-centric study

2019 ◽  
Vol 25 (9) ◽  
pp. 527-537 ◽  
Author(s):  
Sara Stigliani ◽  
Giovanna Orlando ◽  
Claudia Massarotti ◽  
Ida Casciano ◽  
Francesca Bovis ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
Culture Medium ◽  
Embryo Quality ◽  
Culture Media ◽  
Developmental Competence ◽  
Embryo Morphology ◽  
Blastocyst Development ◽  
Mixed Effect ◽  
Non Invasive ◽  
Morphological Criteria

AbstractIn ART, embryo quality evaluation is routinely based on morphological criteria. We previously demonstrated that the mitochondrial DNA (mtDNA)/genomic DNA (gDNA) ratio in culture medium was significantly associated with embryo quality and viability potential. The purpose of this prospective, blinded, multi-centric study was to validate the use of mtDNA/gDNA ratio in Day 3 spent medium as a predictor of human embryo developmental competence. The mtDNA/gDNA ratio was assessed in Day 3 culture media (n=484) of embryos from 143 patients by quantitative PCR. A mixed effect logistic regression model was applied. We found that mtDNA/gDNA ratio in Day 3 culture medium combined with embryo morphology improves the prediction upon blastulation compared to morphology alone (P < 0.0001), independent of patient and cycle characteristics. With regard to routine use in clinics, we evaluated the ability of the novel, combined grading score to improve selection of developmentally competent embryos of a single cohort. Including embryos from 44 patients, the sensibility and specificity of the scoring system based on Day 3 morphological stage were 92% and 13%, respectively. Integration with the culture medium mtDNA/gDNA ratio increased the performance of the method (sensibility: 95%; specificity: 65%). The results of this study suggest the possibility of carrying out a non-invasive evaluation of embryonic mtDNA content through the culture medium. When combined with embryo morphology, it has the potential to help embryologists rank embryos and choose which embryo(s) has the greater development potential, and thus should be transferred on Day 3, among sibling embryos with the same morphological grade.

scholarly journals The Effects of Aquilaria malaccensis Leaves Aqueous Extract on Sperm of Sprague Dawley Rats towards Early Embryogenesis

2020 ◽  
Vol 18 (2) ◽  
Author(s):  
Faridah Ismail ◽  
Azantee Yazmie Abdul Wahab ◽  
Muhammad Lokman Md Isa ◽  
Hussin Muhammad ◽  
Raja Arif Shah Raja Ismail ◽  
...  
Keyword(s):  
Embryo Development ◽  
Embryo Quality ◽  
Early Embryo ◽  
Male Rat ◽  
Embryo Morphology ◽  
Sprague Dawley ◽  
Early Embryo Development ◽  
Aquilaria Malaccensis ◽  
Sprague Dawley Rats

Introduction: Oxidative stress induced by excessive and unopposed levels of reactive oxygen species in male reproductive system results in impaired sperm quality, fertilization capacity and poor embryo development. Our goal is to assess the potential effects of Aquilaria malaccensis (AM) leaves, a plant with strong antioxidant property on early embryo development in vitro and embryo quality following fertilization with cyclophosphamide (CP) exposed rat sperm. Materials and Methods: Twenty four male Sprague Dawley rats were allocated into eight groups of three rats (n = 3): control, CP only (200 mg/kg), AM only (100 mg/kg, 300 mg/kg and 500 mg/kg) and CP + AM (100 mg/kg, 300 mg/kg and 500 mg/kg). Animals were sacrificed after 63 days of treatment and sperm from caudal epididymis were taken for in vitro fertilization (IVF) with oocytes from untreated female. Fertilization, embryo division and embryo morphology were examined at 8 and 48 hours post insemination and compared between groups. Statistical evaluations were performed using Chi-Square test and Fisher’s exact test and p-value <0.05 was considered significant. Results: Administration of AM leave extract at 100 mg/kg/day to normal rats and CP-exposed rats has significantly increased (p><0.05) the fertilization rate, early cleavage rate and embryo quality when compared to CP only treated group. However, other groups showed no significant differences. Conclusion: Overall, the present results indicate the potential of AM leave extract supplementation to improve the fertility and early embryo development in male rat exposed to CP by inhibiting the oxidative processes and scavenging free radicals.>

scholarly journals The invasive and new non-invasive methods of mammalian oocyte and embryo quality assessment: a review

2012 ◽  
Vol 57 (No. 4) ◽  
pp. 169-176 ◽  
Author(s):  
D. Bukowska ◽  
B. Kempisty ◽  
H. Piotrowska ◽  
R. Walczak ◽  
P. Sniadek ◽  
...  
Keyword(s):  
Quality Assessment ◽  
Embryo Quality ◽  
Developmental Potential ◽  
Vitro Fertilization ◽  
Lab On Chip ◽  
New Device ◽  
Non Invasive ◽  
Mammalian Oocyte ◽  
On Chip

&nbsp;The quality of oocytes-embryos can be determined by several techniques, including morphological, molecular, cellular and biochemical ones. The morphological methods of female gamete or embryo quality assessment often use thе following in vitro manipulation procedures such as: in vitro maturation (IVM), in vitro fertilization (IVF) and in vitro embryo production (IVP). However, these methods are highly subjective and the morphological classification of oocytes or embryos is not always compatible with their ability to grow and develop. Additionally, molecular biology methods are objective and present parametric results, which are more or less comparable to the real oocyte-embryo &ldquo;health&rdquo;. Although these techniques enable us to determine markers of oocyte-embryo developmental potential, when applied they lead to destruction of the analysed cells. Therefore, the need still exists to search for new methods that will be highly objective (parametric) and, which is most important, non-invasive. In this review, the morphological and molecular methods of oocyte-embryo quality assessment are presented. Moreover, we described a new system based on microfluidic technology (Lab-on-Chip) which allows the creation of a new device for mammalian oocyte as well as embryo quality evaluation: by using their spectral characterisation following embryo transfer (ET) procedures in the cattle and the pig. &nbsp; &nbsp;

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