Cellular and Molecular Events that Occur in the Oocyte during Prolonged Ovarian Storage in Sheep

For the past two decades, there has been a growing interest in the application of in vitro embryo production (IVP) in small ruminants such as sheep. To improve efficiency, a large number abattoir-derived ovaries must be used, and long distances from the laboratory are usually inevitable when adult animals are used. In that scenario, prolonged sheep ovary transportation may negatively affect oocyte developmental competence. Here, we evaluated the effect of ovary storage time (3, 5, 7, 9, 11 and 13 h) and the medium in which they were transported (TCM199 and saline solution) on oocyte quality. Thus, live/dead status, early apoptosis, DNA fragmentation, reduced glutathione (GSH) and reactive oxygen species (ROS) content, caspase-3 activity, mitochondrial membrane potential and distribution, and relative abundance of mRNA transcript levels were assessed in oocytes. After in vitro maturation (IVM), cumulus cell viability and quality, meiotic and fertilization competence, embryo rates and blastocyst quality were also evaluated. The results revealed that, after 7 h of storage, oocyte quality and developmental potential were significantly impaired since higher rates of dead oocytes and DNA fragmentation and lower rates of viable, matured and fertilized oocytes were observed. The percentage of cleavage, blastocyst rates and cumulus cell parameters (viability, active mitochondria and GSH/ROS ratio) were also decreased. Moreover, the preservation of ovaries in medium TCM199 had a detrimental effect on cumulus cells and oocyte competence. In conclusion, ovary transport times up to 5 h in saline solution are the most adequate storage conditions to maintain oocyte quality as well as developmental capacity in sheep. A strategy to rescue the poor developmental potential of stored oocytes will be necessary for successful production of high-quality embryos when longer ovarian preservation times are necessary.

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P–219 mtDNA content in bovine cumulus cells does not predict oocytés developmental competence

Human Reproduction ◽

10.1093/humrep/deab130.218 ◽

2021 ◽

Vol 36 (Supplement_1) ◽

Author(s):

Á Martíne. Moro ◽

I Lamas-Toranzo ◽

L González-Brusi ◽

A Pérez-Gómez ◽

P Bermejo-Álvarez

Keyword(s):

Cumulus Cell ◽

Cumulus Cells ◽

Oocyte Quality ◽

Blastocyst Stage ◽

Early Embryo ◽

Developmental Competence ◽

Success Rates ◽

Developmental Potential ◽

Mtdna Sequence

Abstract Study question Does cumulus cell mtDNA content correlate with oocyte developmental potential in the bovine model? Summary answer The relative amount of mtDNA content did not vary significantly in oocytes showing different developmental outcomes following IVF What is known already Cumulus cells are closely connected to the oocyte through transzonal projections, serving essential metabolic functions during folliculogenesis. These oocyte-supporting cells are removed and discarded prior to ICSI, thereby constituting an interesting biological material on which to perform molecular analysis aimed to predict oocyte developmental competence. Previous studies have positively associated oocytés mtDNA content with developmental potential in both animal models and women. However, it remains debatable whether mtDNA content in cumulus cells could be used as a proxy to infer oocyte developmental potential. Study design, size, duration Bovine cumulus cells were allocated into three groups according to the developmental potential of the oocyte: 1) oocytes developing to blastocysts following IVF (Bl+Cl+), 2) oocytes cleaving following IVF but arresting their development prior to the blastocyst stage (Bl-Cl+), and 3) oocytes not cleaving following IVF (Bl-Cl-). Relative mtDNA content was analysed in 40 samples/group, each composed by the cumulus cells from one cumulus-oocyte complex (COC). Participants/materials, setting, methods Bovine cumulus-oocyte complexes were obtained from slaughtered cattle and individually matured in vitro (IVM). Following IVM, cumulus cells were removed by hyaluronidase treatment, pelleted, snap frozen in liquid nitrogen and stored at –80 ºC until analysis. Cumulus-free oocytes were fertilized and cultured in vitro individually and development was recorded for each oocyte. Relative mtDNA abundance was determined by qPCR, amplifying a mtDNA sequence (COX1) and a chromosomal sequence (PPIA). Statistical differences were tested by ANOVA. Main results and the role of chance Relative mtDNA abundance did not differ significantly (ANOVA p > 0.05) between the three groups exhibiting different developmental potential (1±0.06 vs. 1.19±0.05 vs. 1.11±0.05, for Bl+Cl+ vs. Bl-Cl+ vs. Bl-Cl-, mean±s.e.m.). Limitations, reasons for caution Experiments were conducted in the bovine model. Although bovine folliculogenesis, monoovulatory ovulation and early embryo development exhibit considerable similarities with that of humans, caution should be taken when extrapolating these data to humans. Wider implications of the findings: The use of molecular markers for oocyte developmental potential in cumulus cells could be used to enhance success rates following single-embryo transfer. Unfortunately, mtDNA in cumulus cells was not found to be a good proxy for oocyte quality. Trial registration number Not applicable

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The Role of MAPK3/1 and AKT in the Acquisition of High Meiotic and Developmental Competence of Porcine Oocytes Cultured In Vitro in FLI Medium

International Journal of Molecular Sciences ◽

10.3390/ijms222011148 ◽

2021 ◽

Vol 22 (20) ◽

pp. 11148

Author(s):

Radek Procházka ◽

Alexandra Bartková ◽

Lucie Němcová ◽

Matej Murín ◽

Ahmed Gad ◽

...

Keyword(s):

Time Course ◽

Cumulus Cell ◽

Cell Communication ◽

Oocyte Quality ◽

Developmental Competence ◽

Control Medium ◽

Developmental Potential ◽

Akt Activation ◽

Expression Of Genes

The developmental potential of porcine oocytes cultured in vitro was remarkably enhanced in a medium containing FGF2, LIF and IGF1 (FLI) when compared to a medium supplemented with gonadotropins and EGF (control). We analyzed the molecular background of the enhanced oocyte quality by comparing the time course of MAPK3/1 and AKT activation, and the expression of genes controlled by these kinases in cumulus-oocyte complexes (COCs) cultured in FLI and the control medium. The pattern of MAPK3/1 activation in COCs was very similar in both media, except for a robust increase in MAPK3/1 phosphorylation during the first hour of culture in the FLI medium. The COCs cultured in the FLI medium exhibited significantly higher activity of AKT than in the control medium from the beginning up to 16 h of culture; afterwards a deregulation of AKT activity occurred in the FLI medium, which was not observed in the control medium. The expression of cumulus cell genes controlled by both kinases was also modulated in the FLI medium, and in particular the genes related to cumulus-expansion, signaling, apoptosis, antioxidants, cell-to-cell communication, proliferation, and translation were significantly overexpressed. Collectively, these data indicate that both MAPK3/1 and AKT are implicated in the enhanced quality of oocytes cultured in FLI medium.

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Neither gonadotropin nor cumulus cell expansion is needed for the maturation of competent porcine oocytes in vitro

Biology of Reproduction ◽

10.1093/biolre/ioab090 ◽

2021 ◽

Author(s):

Bethany K Redel ◽

Lee D Spate ◽

Ye Yuan ◽

Clifton N Murphy ◽

R Michael Roberts ◽

...

Keyword(s):

Assisted Reproductive Technologies ◽

Cumulus Cell ◽

Reproductive Technologies ◽

Oocyte Quality ◽

Blastocyst Stage ◽

Developmental Competence ◽

Oocyte Competence ◽

Key Indicator ◽

Cytoplasmic Competence

Abstract In vitro maturation of oocytes from immature females is widely used in assisted reproductive technologies. Here we illustrate that cumulus cell (CC) expansion, once considered a key indicator of oocyte quality, is not needed for oocytes to mature to the metaphase II (MII) stage and to gain nuclear and cytoplasmic competence to produce offspring. Juvenile pig oocytes were matured in four different media: 1) Basal (−gonadotropins (GN)-FLI); 2) -GN + FLI (supplement of FGF2, LIF, and IGF1); 3) + GN-FLI; 4) + GN + FLI. There was no difference in maturation to MII or progression to the blastocyst stage after fertilization of oocytes that had been matured in -GN + FLI medium and oocytes matured in +GN + FLI medium. Only slight CC expansion occurred in the two media lacking GN compared to the two where GN was present. The cumulus-oocytes-complexes (COC) matured in +GN + FLI exhibited the greatest expansion. We conclude that FLI has a dual role. It is directly responsible for oocyte competence, a process where GN are not required, and, when GN are present, it has a downstream role in enhancing CC expansion. Our study also shows that elevated phosphorylated MAPK may not be a necessary correlate of oocyte maturation and that the greater utilization of glucose by COC observed in +GN + FLI medium probably plays a more significant role to meet the biosynthetic needs of the CC to expand than to attain oocyte developmental competence. Gene expression analyses have not been informative in providing a mechanism to explain how FLI medium enhances oocyte competence without promoting CC expansion.

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205 CUMULUS CELL MORPHOLOGY BEFORE AND AFTER IN VITRO MATURATION AS AN INDICATOR OF PORCINE OOCYTE DEVELOPMENTAL COMPETENCE

Reproduction Fertility and Development ◽

10.1071/rdv22n1ab205 ◽

2010 ◽

Vol 22 (1) ◽

pp. 260

Author(s):

M. Bertoldo ◽

P. K. Holyoake ◽

G. Evans ◽

C. G. Grupen

Keyword(s):

Cell Morphology ◽

In Vitro Maturation ◽

Cumulus Cell ◽

Cumulus Cells ◽

Oocyte Quality ◽

Developmental Competence ◽

Oocyte Developmental Competence ◽

Before And After ◽

Follicle Size

Effective in vitro maturation (IVM) is essential for successful in vitro embryo production. The morphology of the cumulus investment before and after IVM may be a useful noninvasive indicator of oocyte quality. In pigs, oocyte developmental competence is reduced during the summer months. The aim of this study was to determine whether the morphology of cumulus-oocyte complexes (COC) before and after IVM are associated with oocyte quality, using COC collected from small and large follicles in summer and winter as models of poor and good oocyte quality. Ovaries were collected from sows slaughtered 4 days after weaning. The COC recovered from small (3-4 mm) and large (5-8 mm) antral follicles were morphologically graded and parthenogenetically activated following IVM during winter (n = 1419; 10 replicates) and summer (n = 2803; 10 replicates). Grade 1 and 2 COC had >2 layers of compact cumulus cells and a homogenous cytoplasm. Grade 3 COC were either partially or fully denuded, had a heterogeneous cytoplasm, or were vacuolated or dark in color. Grade 4 COC had expanded cumulus cells. Cumulus expansion was also assessed subsequent to IVM. The COC recorded as having a cumulus expansion index (CEI) of 1 had the poorest expansion with no detectable response to IVM, whereas those with a CEI of 4 had the greatest amount of expansion, including that of the corona radiata. Data were analyzed using a generalized linear mixed model in GenStat® (release 10, VSN International, Hemel Hempstead, UK). There was an effect of follicle size for Grade 1 COC, with COC from large follicles in both seasons yielding better quality COC (P < 0.05). The proportion of COC in Grade 2 was higher in small follicles during winter compared with large follicles, but there were no differences between follicle sizes during summer (P < 0.05). The proportion of COC with CEI 1 was highest in COC from small follicles during summer (P < 0.05). The proportion of COC from large follicles with CEI 2 was higher during summer compared with winter (P < 0.05). There were no seasonal or follicle size effects on COC with CEI 3 or 4 (P > 0.05). The proportion of oocytes that developed to blastocysts was greater in winter than in summer (39.06% ± 5.67 v. 22.27% ± 4.01; P < 0.05). Oocytes derived from large follicles had a greater ability to form blastocysts compared with those from small follicles (37.13% ± 5.65 v. 23.32% ± 4.56; P < 0.06). Morphological assessment of cumulus cells before and after IVM may be a useful tool to evaluate the effects of follicle size on oocyte developmental competence. However, the results of the present study indicate that cumulus cell morphology is not a good indicator of the effect of season on oocyte developmental competence.

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170 INFLUENCE OF OOCYTE QUALITY AND DIFFERENT MEDIA SUPPLEMENT ON IN VITRO MATURATION, CLEAVAGE, AND EMBRYO DEVELOPMENT OF BUFFALO (BUBALUS BUBALIS) OOCYTES

Reproduction Fertility and Development ◽

10.1071/rdv26n1ab170 ◽

2014 ◽

Vol 26 (1) ◽

pp. 199

Author(s):

M. M. Waheed ◽

K. H. El-Shahat ◽

A. M. Hammam

Keyword(s):

Embryo Development ◽

In Vitro Maturation ◽

Cumulus Cell ◽

Oocyte Quality ◽

Bubalus Bubalis ◽

Poor Quality ◽

Developmental Competence ◽

Control Group ◽

Excellent Quality

A series of 4 factorial-arranged experiments were conducted to study the effect of oocyte quality and different in vitro maturation (IVM) media supplements on IVM, cleavage, and embryo development of buffalo (Bubalus bubalis) oocytes. Buffalo ovaries were collected at a local abattoir in a warm (32–35°C) saline (0.9% NaCl), and oocytes were aspirated using an 18-gauge needle. In experiment 1, oocytes (n = 320) were classified according to the number of cumulus cell layers and morphology of ooplasm as excellent, good, or fair. Oocytes were cultured for IVM, fertilization, and embryo culture (IVMFC) in TCM-199 + 10% FCS. In experiment 2, excellent quality oocytes (n = 237) were subjected to IVM in TCM-199 enriched with either 10% FCS or oestrous buffalo serum (EBS; 20–40 pg mL–1) and then fertilized using frozen–thawed buffalo semen capacitated in Bracket and Oliphant's (BO) medium containing heparin (0.02 mg mL–1) and sodium caffeine benzoate (3.89 mg mL–1). In experiment 3, oocytes (n = 290) were classified into 2 groups; Group 1, without gonadotropins, served as a control; Group 2, in which IVM medium was supplemented with 20 IU mL–1 equine chorionic gonadotropins (eCG). Experiment 4 was carried out to examine the suitable capacitating agent added to BO medium, either heparin or caffeine or both (n = 210 fertilized oocytes). In all experiments (multiple replicates), oocytes (2–6 mm in diameter) were kept at 39°C under 5% CO2 for IVMFC and examined several times for cleavage and embryo development (morula and blastocyst). Statistical analysis was carried out using Chi-squared test. Excellent and good quality oocytes produced a higher (P < 0.05) maturation, cleavage, and morula development rates than poor quality oocytes (70% and 65% v. 33.3%), (50% and 46.2% v. 25%), and (42.9% and 33.3% v. 10%), respectively. Blastocyst production rate was also higher (P < 0.05) for excellent compared with good quality oocytes (28.6% v. 16.7%, respectively). In experiment 2, the IVM and cleavage rates were significantly higher (P < 0.05) in IVM medium plus 10% EBS than those cultured in 10% FCS (73% v. 45% and 50% v. 33.3%, respectively). In experiment 3, the addition of eCG to maturation medium increased (P < 0.05) developmental competence of buffalo oocytes (IVMFC) compared with control medium (16% v. 4%). In experiment 4, the addition of heparin together with caffeine to BO medium produced significantly higher (P < 0.05) cleavage and embryo developmental rates compared with heparin or caffeine alone (56.3% v. 33.3% and 35.7%, respectively; 22.2% v. 10% and 8%, respectively). In conclusion, excellent quality oocytes cultured in IVM medium supplemented with either EBS or eCG and fertilized with capacitated buffalo spermatozoa in BO medium enriched with heparin and caffeine progressively enhanced developmental competence of buffalo oocytes.

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020. IN VITRO MATURATION OF FARM ANIMALS OOCYTES: A USEFUL TOOL FOR INVESTIGATING THE MECHANISMS LEADING TO FULL TERM DEVELOPMENT

Reproduction Fertility and Development ◽

10.1071/srb09abs020 ◽

2009 ◽

Vol 21 (9) ◽

pp. 7

Author(s):

F. Gandolfi ◽

T. Brevini

Keyword(s):

Large Body ◽

Genetic Material ◽

Oocyte Quality ◽

Full Term ◽

Farm Animals ◽

Ongoing Research ◽

Developmental Potential ◽

Oocyte Competence ◽

Amount Of Knowledge

Due to logistical and economical reasons assisted reproduction of domestic animals has been based mostly on the use of oocytes isolated from ovaries collected at the slaughterhouse. In order to propagate valuable or rare genetic material, perform somatic cell nuclear transfer, generate genetically modified animals it was essential to obtain fully competent oocytes that would allow full term development of the in vitro produced embryos. Such demanding need has soon made clearly evident the crucial role played by oocyte quality, how easy it is to compromise its developmental potential and the fact that it is impossible to restore it once it has been lost. Almost three decades after the first bovine, sheep, goat, horse and pig in vitro generated offsprings were born, a large body of information has accumulated on the mechanisms regulating oocyte competence and on how the latter may be preserved during all the required manipulations. The amount of knowledge is far from being complete and many laboratories are actively working to further expand it. In this review we will highlight the aspects of the ongoing research in which we have been actively involved.

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Failure to launch: aberrant cumulus gene expression during oocyte in vitro maturation

Reproduction ◽

10.1530/rep-16-0426 ◽

2017 ◽

Vol 153 (3) ◽

pp. R109-R120 ◽

Cited By ~ 16

Author(s):

Hannah M Brown ◽

Kylie R Dunning ◽

Melanie Sutton-McDowall ◽

Robert B Gilchrist ◽

Jeremy G Thompson ◽

...

Keyword(s):

Gene Expression ◽

Cell Metabolism ◽

Cumulus Cell ◽

Cumulus Cells ◽

Developmental Competence ◽

Oocyte Competence ◽

Oocyte Developmental Competence ◽

Growth Factor Signalling

In vitro maturation (IVM) offers significant benefits for human infertility treatment and animal breeding, but this potential is yet to be fully realised due to reduced oocyte developmental competence in comparison with in vivo matured oocytes. Cumulus cells occupy an essential position in determining oocyte developmental competence. Here we have examined the areas of deficient gene expression, as determined within microarrays primarily from cumulus cells of mouse COCs, but also other species, between in vivo matured and in vitro matured oocytes. By retrospectively analysing the literature, directed by focussing on downregulated genes, we provide an insight as to why the in vitro cumulus cells fail to support full oocyte potential and dissect molecular pathways that have important roles in oocyte competence. We conclude that the roles of epidermal growth factor signalling, the expanded extracellular matrix, cumulus cell metabolism and the immune system are critical deficiencies in cumulus cells of IVM COCs.

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hCG Priming Before Ovary Collection Increasing The Oocyte Quality In The Domestic Cat

Acta Veterinaria Indonesiana ◽

10.29244/avi...123-126 ◽

2021 ◽

pp. 123-126

Author(s):

Karisma Mardatillah ◽

Rini Widyastuti ◽

Diah Nugrahani Pristihadi ◽

Wahyudin ◽

Sigit Prastowo ◽

...

Keyword(s):

Oocyte Quality ◽

Domestic Cat ◽

Developmental Competence ◽

Control Group ◽

Oocyte Competence ◽

Slicing Method ◽

Determining Factor

Oocyte competence is a determining factor that influences the embryo development. Embryos produced in vitro have a reduced developmental competence than embryos produced in vivo. Therefore, human Chorionic Gonadotropin (hCG) injection was carried out to improve the quality of the oocytes. The objective of this study was to evaluate the effect of ovarian stimulation with hCG before ovary collection on oocyte quality in the domestic cat. Oocyte donors were either 1) treated with a single dose of 200 IU hCG four days before ovary collection (hCG group), or, 2) no treatment before ovary collection (control group). The oocytes were collected by the slicing method. Immature cumulus oophorus complexes (COCs) from both groups were pooled and matured in vitro for 24-26 hours. Then mature oocytes were fertilized with epididymal sperm and cultured in vitro for seven days. The results study showed that the number of the dominant follicle (DF) and the number of COCs in the hCG group was higher than the control group in right and left ovaries (p<0.05). The morulae and blastocyst rates from cleavage embryos were 88% and 75%, respectively. These results demonstrate that hCG priming of oocytes donors before ovary collection improve oocyte quality.

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156 EFFECTS OF MORPHOLOGY TYPE OF POLAR BODY ON PORCINE OOCYTE QUALITY AND DEVELOPMENTAL POTENTIAL AFTER IN VITRO FERTILIZATION

Reproduction Fertility and Development ◽

10.1071/rdv26n1ab156 ◽

2014 ◽

Vol 26 (1) ◽

pp. 192

Author(s):

L. Cai ◽

E. Kim ◽

S. U. Hwang ◽

J. D. Yoon ◽

Y. Jeon ◽

...

Keyword(s):

Polar Body ◽

Oocyte Quality ◽

Developmental Competence ◽

Penicillin G ◽

Cleavage Rate ◽

Developmental Potential ◽

Vitro Fertilization ◽

First Polar Body

Evaluation of morphology of first polar body (1st PB) could be a method for the oocyte's quality and developmental competence. The developmental potential of oocyte with fragmented PB after in vitro maturation (IVM) is a controversial issue. The aim of this study is to investigate the effects of PB morphology type on oocyte quality and developmental competence after IVF. Porcine ovaries were obtained from prepubertal gilts at a local slaughterhouse and transported to the laboratory within 2 h in physiological saline supplemented with 100 IU mL–1 penicillin G and 100 mg mL–1 streptomycin sulfate. The cumulus–oocyte complexes (COC) were aspirated using an 18-gauge needle attached to a 10-mL disposable syringe from superficial follicles 3 to 6 mm in diameter followed by IVM. After IVM, oocytes were classified into 3 types as follows, oocytes with normal PB (A type), oocytes with a little of fragmented PB (B type), and oocytes with separated 2 PBs (C type), respectively. As classification of PB types, we analysed the distribution ratio of each PB type after IVM, and then performed IVF for analysis of fertilization rate and developmental potential. The ratio of oocyte with A type (73%) was significantly (P < 0.05) higher than that of B type (24.5%) or C type (2.5%) after IVM. Only mature oocytes were selected from A and B type and were subjected to IVF because of a small number of oocytes with C type. In the IVF experiment, the efficiency of monospermy and fertilization were significantly higher in oocytes of A type (46.7%) than those of type B (20.0%). The cleavage rate of oocytes with A type (63.9%) was significantly (P < 0.05) higher than the oocytes with B type (43.8%). Embryonic developmental competence to the blastocyst stage after IVF was significantly (P < 0.05) higher in the A-type oocytes (26.3%) than in the B-type oocytes (16.9%). The levels of glutathione and reactive oxygen species were not affected by the morphological classification of the PB. In summary, these results suggest that polar body morphology could be a marker of oocyte quality after IVM. We are currently studying gene expression of each oocytes and blastocysts. This work was supported, in part, by a grant from the Next-Generation BioGreen 21 Program (No. PJ00956901), Rural Development Administration, and the National Research Foundation of Korea Grant funded by the Korean Government (NRF-2012R1A1A4A01004885, NRF-2013R1A2A2A04008751), Republic of Korea.

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Evaluation of bovine zona pellucida characteristics in polarized light as a prognostic marker for embryonic developmental potential

Reproduction ◽

10.1530/rep-10-0471 ◽

2011 ◽

Vol 141 (6) ◽

pp. 779-787 ◽

Cited By ~ 13

Author(s):

M Koester ◽

A Mohammadi-Sangcheshmeh ◽

M Montag ◽

F Rings ◽

T Schimming ◽

...

Keyword(s):

Zona Pellucida ◽

Polarized Light ◽

Oocyte Quality ◽

Developmental Competence ◽

Blue Staining ◽

Developmental Potential ◽

Preimplantation Embryo Development ◽

Intensity Parameters ◽

Bovine Oocytes

It has previously been demonstrated that zona pellucida imaging of human oocytes using polarized light microscopy is a clinically applicable method for the noninvasive assessment of oocyte quality. This study was designed to investigate whether zona pellucida characteristics of bovine oocytes and zygotes in polarized light may similarly serve as a useful marker for developmental competence in bovine reproductive biotechnologies. Zona birefringence intensity parameters of 2862 oocytes/zygotes were objectively evaluated with an automatic analysis system and correlated with oocyte/zygote quality. In detail, immature oocytes of good quality assessed with brilliant cresyl blue staining showed significantly lower zona birefringence than poor-quality counterparts (P<0.001). Afterin vitromaturation and classification according to maturational status, the birefringence intensity parameters were significantly different in those oocytes that reached metaphase II compared with arrested stages (P<0.001). Following either parthenogenetic activation or IVF with subsequentin vitroculture in a well-of-the-well system until day 9, superior development as determined by cleavage, blastocyst formation, and hatching ability was associated with lower zona birefringence intensity parameters. When early zygote-stage embryos were selected and assorted in groups based on zona birefringence (high/medium/low), the group of embryos derived from high-birefringence zygotes displayed a significantly compromised developmental potential compared with low-birefringence zygotes. These results clearly show that developmentally competent bovine oocytes/zygotes exhibit lower zona birefringence intensity parameters. Therefore, birefringence imaging of zona pellucida is a suitable technique to predict bovine preimplantation embryo development.

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