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Adenovirus is a kind of non-enveloped,double-stranded DNA virus. As a member of the mammalian adenoviruses of the Adenoviridae family, porcine adenovirus causes gastrointestinal disease in piglets. In this study, the modified adenovirus was manipulated to carry a green fluorescence EGFP marker. The modified adenovirus was added to medium199 for co-incubation or microinjected into the cytoplasm of porcine oocytes. The effect of adenovirus on the first polar body extrusion was not significant during porcine oocyte maturation. Our data demonstrated the zona pellucida plays a vital role in porcine oocytes being resistant to modified adenovirus. Additionally, the results suggested that oocytes protect themselves from nonself substances.

The present study was designed to investigate the comprehensive function of maternal factors of primordial germ cell 7 (PGC7) and POU5F1—POU class 5 homeobox 1 (OCT4), as well as the epige...

The work aimed (Experiment I) to compare commercial representations of porcine follicle stimulating hormone (FSH, Pluset® vs. Folltropin®) in concentration (10 ?g/mL) and time (24 h) standard (more used in protocols of in vitro maturation, IVM); (Experiment II) to evaluate the best incubation time (6 h vs. 16 h vs. 24 h) and, (Experiment III) analyze varying concentrations (1.0 ?g/mL vs. 2.5 ?g/mL vs. 10.0 ?g/mL) of representations of FSH on the IVM of bovine oocytes. Thus, oocytes were recovered and submitted to IVM under appropriate conditions. After the IVM, oocytes were evaluated for expansion of cumulus cells (CCs), presence of the first polar body (1PB) and metaphase plate (MII). All the data were analyzed by the Fisher exact test (P < 0.05). Initially, in Experiment I, no difference (P > 0.05) was observed in maturation rates of the oocytes incubated with FSH Pluset® or Folltropin®, assessed by expansion of CCs (97.6% vs. 94.3%), presence of 1PB (76.6% vs. 69.4%) and MII (70.0% vs. 68.6%). In Experiment II, when the incubation time with FSH was evaluated, both Pluset® as Folltropin® showed lower rate of expansion of CCs when they were present only in the first 6 h of IVM. As for presence of 1PB, differences were observed in relation to Pluset® while Folltropin® showed similar results in all incubation times. Regarding the MII, no difference was observed between the incubation times with FSH Pluset® and Folltropin®. In Experiment III, no difference was observed in the expansion of CCs, presence of 1PB and MII for concentrations evaluated FSH Pluset® and Folltropin®. Therefore, the FSH Pluset® and Folltropin® have the same efficiency in IVM of bovine oocytes. Regarding the incubation time, it is recommended to maintain FSH (Pluset® or Folltropin®) throughout the period of IVM, since there was no difference in the results of presence of MII. Furthermore, the concentration of 1.0 ?g/mL of FSH Pluset® and Folltropin® is as effective as 10 ?g/mL and can therefore be used for IVM of oocytes.

Mammalian oocytes carry specific nongenetic information, including DNA methylation to the next generation, which is important for development and disease. However, evaluation and manipulation of specific methylation for both functional analysis and therapeutic purposes remains challenging. Here, we demonstrate evaluation of specific methylation in single oocytes from its sibling first polar body (PB1) and manipulation of specific methylation in single oocytes by microinjection-mediated dCas9-based targeted methylation editing. We optimized a single-cell bisulfite sequencing approach with high efficiency and demonstrate that the PB1 carries similar methylation profiles at specific regions to its sibling oocyte. By bisulfite sequencing of a single PB1, the methylation information regarding agouti viable yellow ( A vy )-related coat color, as well as imprinting linked parthenogenetic development competency, in a single oocyte can be efficiently evaluated. Microinjection-based dCas9-Tet/Dnmt–mediated methylation editing allows targeted manipulation of specific methylation in single oocytes. By targeted methylation editing, we were able to reverse A vy -related coat color, generate full-term development of bimaternal mice, and correct familial Angelman syndrome in a mouse model. Our work will facilitate the investigation of specific methylation events in oocytes and provides a strategy for prevention and correction of maternally transmitted nongenetic disease or disorders.

The aim of this study was to evaluate the effect of three different incubation times on in vitro maturation of domestic cat oocytes. Thus, ovaries (n = 42) were submitted to slicing procedure and the oocytes recovered were classified; only good quality oocytes (Grade I and II) underwent in vitro maturation for three different periods (24 vs. 30 vs. 36 h) in supplemented TCM-99 medium. After, oocytes were evaluated for cumulus cell expansion and presence of the first polar body. After six replicates (7 ± 1,7 ovaries per replicate), a total of 334 viable oocytes were recovered. Differences (p

The combination of in vitro maturation (IVM) techniques and oocyte vitrification (OV) could increase the number of useful oocytes in different types of patients. IVM and subsequent OV is the most widely used clinical strategy. Would the results improve if we reverse the order of the techniques? Here, we evaluated survival, in vitro maturation, time to extrude the first polar body (PB), and the metaphase plate configuration of human prophase I (GV) oocytes before or after their vitrification. Specific, 195 GV oocytes from 104 patients subjected to controlled ovarian stimulation cycles were included. We stablished three experimental groups: GV oocytes vitrified and IVM (Group GV-Vit), GV oocytes IVM and vitrified at MII stage (Group MII-Vit), and GV oocytes IVM (Group not-Vit). All of them were in vitro matured for a maximum of 48 h and fixed to study the metaphase plate by confocal microscopy. According to our results, the vitrification of immature oocytes and their subsequent maturation presented similar survival, maturation, and metaphase plate conformation rates, but a significantly higher percentage of normal spindle than the standard strategy. Additionally, the extension of IVM time to 48 h did not seem to negatively affect the oocyte metaphase plate configuration. This research was funded by Department of Biotechnology of the University of Alicante (VIGROB-186).

Cell microinjection is a technique of precise delivery of substances into cells and is widely used for studying cell transfection, signaling pathways, and organelle functions. Microinjection of the embryos of zebrafish, the third most important animal model, has become a very useful technique in bioscience. However, factors such as the small cell size, high cell deformation tendency, and transparent zebrafish embryo membrane make the microinjection process difficult. Furthermore, this process has strict, specific requirements, such as chorion softening, avoiding contacting the first polar body, and high-precision detection. Therefore, highly accurate control and detection platforms are critical for achieving the automated microinjection of zebrafish embryos. This article reviews the latest technologies and methods used in the automated microinjection of zebrafish embryos and provides a detailed description of the current developments and applications of robotic microinjection systems. The review covers key areas related to automated embryo injection, including cell searching and location, cell position and posture adjustment, microscopic visual servoing control, sensors, actuators, puncturing mechanisms, and microinjection.

Chromosome segregation in fertilized eggs from triploid Pacific oysters, following inhibition of the first polar body (PB1), was studied with acetic orcein staining techniques. To block the release of PB1, fertilized eggs were treated with 0.5 mg/l of cytochalasin B (CB). Four types of segregation were observed, namely, "tripolar segregation" (54.5%), "united bipolar segregation" (12%), "separated bipolar segregation" (2.5%), and "incomplete united bipolar segregation" (4%). The remaining 23% could not be classified because of chromosome disorganization, but appeared to be variants of the above. It seemed clear that the predominant pattern that gave rise to tetraploids was united bipolar segregation, although certain separated bipolar segregations might also lead to the formation of tetraploids. The sequential events of meioses observed in CB-treated eggs are described. The asynchrony of meiotic events and possible mechanisms for the various types of chromosome segregation are discussed.

Ban is an endangered miniature pig breed in Vietnam. This study aimed to set up an in vitro embryo production (IVP) system for this breed. Ban's epididymal sperm concentration (1240 ± 35 × 10(6) /mL) was lower (P 2 mm follicles). After in vitro maturation, the percentages of oocytes with expanded cumulus cells and the first polar body (matured oocytes) were not different among Ban, hormone-stimulated Ban and Landrace. The percentages of two-cell embryos and morulae derived from oocytes collected from three sources did not differ. However, the rate of blastocysts derived from oocytes in non-stimulated Ban (4.0 ± 3.8%) was lower (P

SummaryPrevious studies have reported that the first polar body (PB1) morphology reflects embryo development competence, but the effects of PB1 on porcine embryo development remain unknown. This study aims to determine whether the ability of porcine embryo development is related to oocytes’ PB1 in vitro. The distribution of type II cortical granules (CGs) of porcine matured oocytes in grade B PB1 is significantly greater compared with those in grades A and C PB1 (71.43% versus 52.46% and 50%; P < 0.05). The ratio of porcine parthenogenetic blastocysts and the mean cell number in each blastocyst in the group with grade B PB1 is significantly greater than that with grades A and C PB1 (30.81% vs. 19.02% and 15.15%; P < 0.05) and (36.67 versus 24.67, 28.67; P < 0.05), and no significant differences are found in the embryo cleavage for all groups (79.75%, 84.30%, and 78.18% in grades A, B, and C PB1; P > 0.05). The acetylation level of porcine embryos in the group with grade B PB1 is significantly greater compared with those in the other groups (P < 0.05), and is almost 2.5 times higher than that in grade A. Therefore, porcine oocytes with PB1 in grade B are more competitive in cytoplasmic maturation and further embryo development in vitro.

The capability to precisely rotate cells and other micrometer-sized biological samples is invaluable in biomedicine, bioengineering, and biophysics. We propose herein a novel on-chip cell rotation method using acoustic microstreaming generated by oscillating asymmetrical microstructures. When the vibration is applied to a microchip with our custom-designed microstructures, two different modes of highly localized microvortices are generated that are utilized to precisely achieve in-plane and out-of-plane rotational manipulation of microbeads and oocytes. The rotation mechanism is studied and verified using numerical simulations. Experiments of the microbeads are conducted to evaluate the claimed functions and investigate the effects of various parameters, such as the frequency and the driving voltage on the acoustically induced flows. Accordingly, it is shown that the rotational speed and direction can be effectively tuned on demand in single-cell studies. Finally, the rotation of swine oocytes is involved as further applications. By observing the maturation stages of M2 after the exclusion of the first polar body of operated oocytes, the proposed method is proved to be noninvasive. Compared with the conventional approaches, our acoustofluidic cell rotation approach can be simple-to-fabricate and easy-to-operate, thereby allowing rotations irrespective of the physical properties of the specimen under investigation.

Background & objectives: The major cause of fertilisation failure after ICSI is failure of the oocyte to initiate the biochemical processes necessary for activation. This inability could be ascribed to cytoplasmic immaturity of those gametes even if they had reached nuclear maturity. The activation of a mature oocyte is characterised by release from metaphase II (MII) arrest and extrusion of the second polar body, followed by pro-nuclear formation. The aim of this study was to evaluate the fate of in vitro matured (IVM) metaphase I (MI) oocytes subjected to intracytoplasmic sperm injection (ICSI) at different time intervals after extrusion of the first polar body (1PB) in in vitro fertilization (IVF) cycles. Methods: A total of 8030 oocytes were collected from 1400 ICSI cycles, 5504 MII at the time of cumulus retrieval. Four hundred eight metaphase II (MII) (27.1%) matured to MII after in vitro culture for 2-26 h and 5389 sibling MII in the moment of oocyte denudation were injected. On the other hand, 49 ICSI cycles containing only MI oocytes at retrieval were injected at three different time intervals after reaching the MII. The intervals were as follows: 2-6 h (n=10), 8-11 h (n=4) and 23-26 h (n=10). Fertilization and development potential were evaluated in both studies. Results: Fertilization, embryo cleavage and quality were significantly lower in IVM MI compared to MII at time of denudation. Pregnancy rate was higher in group MII. Pregnancy was achieved in three embryo transfers when ICSI was performed within 2-6 h (group I) and 8-11 h (group II) after PB extrusion. One pregnancy was obtained in group I and a healthy neonate was born. Interpretation & conclusions: Immature oocytes from women whose ovaries have been stimulated could be matured, fertilized by ICSI, cleaved in vitro and to give rise to a live birth. However, the developmental competence of embryos derived from immature oocytes is reduced, compared with sibling in vivo matured oocytes. Further, human IVM oocytes need between 2-6h after the 1PB extrusion to complete its maturation.

This study was undertaken to investigate whether complete removal or downsizing of the cumulus cell layers in germinal vesicle (GV)-stage bovine cumulus-oocyte complexes (COCs) can improve blastocyst development rate following Cryotop vitrification. Downsized COCs (196 μm in mean diameter) and denuded oocytes (141 μm in mean diameter) were prepared by vortex-mixing of full-sized COCs (330 μm in mean diameter) retrieved from abattoir-derived ovaries. Nuclear maturation rates, assessed by the first polar body extrusion, after vitrification and the subsequent 22-h IVM were comparable (61.9–62.9%). Approximately one-third (30.5–31.2%) of the matured oocytes derived from the downsized COCs could develop into high quality blastocysts after 6-h IVF and 8-d IVC, while 13.4 and 23.7% of the matured oocytes derived from denuded oocytes and full-size COCs reached to the blastocysts, respectively. Cytoplasmic lipid droplets of matured oocytes in vitrification group were more clustered with decreased number and increased size of the droplets, when compared to those in fresh control group. However, individual oocyte culture in well-of-the well system suggested that change of lipid droplet distribution in the matured oocytes had no adverse effect on their subsequent developmental competence up to the blastocyst stage. In conclusion, Cryotop vitrification of downsized GV-stage bovine COCs allowed blastocyst yields as high as >30%.

Objective To evaluate the dynamics of the nuclear maturation (NM) of in vitro-matured (IVM) oocytes and to determine the most favorable duration of meiosis II (MII) arrest in relation to the normal activation response. Design Experimental. Setting University-affiliated infertility clinic. Patient(s) Donated immature germinal vesicle oocytes (GV). Intervention(s) The GV underwent spontaneous IVM and the dynamics of NM studied by real-time monitoring. The IVM oocytes were parthenogenetically activated at different MII arrest points and their response assessed. Main Outcome Measure(s) Moment of GV breakdown; extrusion of the first polar body; duration of MI and MII arrest; activation rate (AR) and type. Result(s) Two GV populations—early (E-IVM, 18.4 ± 2.7 hours) and late (L-IVM, 26.3 ± 3.8 hours) maturing—were defined according to the time required for extrusion of the first polar body. Significantly more E-IVM than L-IVM exhibited a normal activation response (61.3% vs. 34.6%), but AR were similar (average, 88.6%) in both groups. Duration of the GV stage differed between the two groups, but MI arrest (14.0 ± 0.3 hours) was constant. The E-IVM arrested at MII for at least 4.3 hours displayed significantly lower AR and similar normal activation rates (61.3%) to E-IVM arrested for a shorter time (83.9% vs. 100%). The L-IVM displayed a similar AR (80.8%), but lower normal activation rates than E-IVM (34.6%), regardless of when activation took place. Conclusion(s) The success of IVM depends on the NM timing rather than on the length of MII arrest.

Fil: Rodríguez, María Belén. Universidad de Buenos Aires. Facultad de Agronomía. Laboratorio de Biotecnología Animal; Argentina Fil: Rodríguez, María Belén. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Gambini, Andrés. Universidad de Buenos Aires. Facultad de Agronomía. Laboratorio de Biotecnología Animal; Argentina Fil: Gambini, Andrés. Consejo Nacio al de Investigaciones Científicas y Técnicas; Argentina Fil: Clérico, Gabriel José. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Clérico, Gabriel José. Pontificia Universidad Católica Argentina. Facultad de Ingeniería y Ciencias Agrarias. Laboratorio de Biotecnología y Reproducción Animal; Argentina Fil: Ynsaurralde Rivolta, Amada Eugenia. Instituto Nacional de Tecnología Agropecuaria; Argentina Fil: Briski, Olinda. Universidad de Buenos Aires. Facultad de Agronomía. Laboratorio de Biotecnología Animal; Argentina Fil: Briski, Olinda. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Largel, Hernan. El Palenque Embriones Equine Embryo Transfer Center; Argentina Fil: Sansiñena, Marina Julia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Sansiñena, Marina Julia. Pontificia Universidad Católica Argentina. Facultad de Ingeniería y Ciencias Agrarias. Laboratorio de Biotecnología y Reproducción Animal; Argentina Fil: Salamone, Daniel F. Universidad de Buenos Aires. Facultad de Agronomía. Laboratorio de Biotecnología Animal; Argentina Fil: Salamone, Daniel F. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Abstract: Assisted reproduction techniques (ARTs) have become widespread in the equine breeding industry. In particular, the combination of oocyte recovery from live mares followed by IVM and intracytoplasmic sperm injection (ICSI) has increased markedly among the ARTs used with valuable or low-fertility animals. There is currently no consensus among research groups regarding the optimal oocyte maturation period to produce high-quality embryos. In this study, we report the maturation dynamics of equine oocytes at different time points, from 20 to 40 h (Experiment 1). In addition, in Experiment 2, equine ICSI blastocysts were produced from oocytes that exhibited early (up to 24 h) or late (28–30 h) extrusion of the first polar body (PB). Blastocyst rates and diameter were recorded and embryo quality was assessed by analysing the number of apoptotic cells and Yes-associated protein 1 (YAP1) expression. By 20 h of IVM, 42% of oocytes were mature, and the remaining oocytes matured within the next 17 h of IVM. Although no differences were found in cell apoptosis or the number of YAP1-positive cells between groups exhibiting early and late PB extrusion, embryos from the early group (Group I) exhibited an improved total cell number and blastocyst rate compared to embryos from the late group (Group II) (18.60% vs 10.17% respectively).

In this study, the effects of dibutyryl cyclic AMP (dbcAMP) on the preservation of porcine oocytes were investigated. Oocytes were preserved for 24 h or 48 h in modified NCSU37 supplemented with or without 1 mM dbcAMP. After the preservation, 98.3% and 95.2% of oocytes incubated with dbcAMP for 24 h and 48 h, respectively, were at the germinal vesicle stage, whereas about a half of the oocytes incubated without dbcAMP underwent germinal vesicle break down. These preserved oocytes were cultured for 46 h. The in vitro maturation (IVM) rates of oocytes preserved with dbcAMP for 24 h (84.0%) and 48 h (55.3%) were significantly higher than those of oocytes preserved without dbcAMP (48.0% and 21.6%, respectively). IVM oocytes with a visible first polar body were selected, fertilized in vitro (IVF), and cultured for five days. The rate of oocytes preserved for 24 h with dbcAMP and developed to the blastocyst stage after IVF was 21.4% and did not differ from that of oocytes without preservation (23.8%); however, the blastocyst development rate significantly decreased for oocytes preserved with dbcAMP for 48 h (5.2%). These results indicate that dbcAMP is effective for the preservation of porcine oocytes for 24 h without decreasing their developmental ability after IVF.

Beside cooling/warming rates and composition of vitrification solution, developmental stage of immature oocytes may also affect their vitrification outcome. The aim of the present study was to evaluate the selection effect of developmentally competent immature bovine oocytes by Brilliant Cresyl Blue (BCB) on maturity of oocytes after vitrification. Oocytes were obtained from slaughterhouse ovaries. Only oocytes with 4-5 layers of cumulus cells and homogenous cytoplasm were used. After exposure to BCB stain, immature oocytes were divided into colored (BCB+) and colorless (BCB-) cytoplasm groups. Immature oocytes were equilibrated in VS1 (7.5 Ethylene Glycol (EG)+7.5% DMSO) for 10-12 min and then exposed to VS2 (15% EG+ 15% DMSO+0.5M sucrose) for 1 min. Thereafter, oocytes were loaded on Cryotop and directly plunged into liquid nitrogen. After warming, oocytes were examined for presence of polar body and nuclear maturity. Higher number of oocytes in BCB+group extruded first polar body in comparison with other vitrified groups but not significantly (p>0.05). Compared to the BCB- oocytes, there was significantly lower percentage of degeneration for BCB+oocytes (p

The ability to rescue gametes from endangered or wildlife species and to subsequently produce viable embryos holds tremendous potential as a means to increase the population size of endangered or wildlife species. The objective of this study was to assess the meiotic and developmental competence of oocytes recovered from postmortem ovaries of the Indian blackbuck. Oocytes collected from the ovaries of dead blackbucks were allowed to mature in vitro and then tested for developmental potential by activation with ionomycin followed by treatment with 6-dimethylaminopurine. The average number of oocytes recovered per ovary was 10.9, and recovery of the oocytes did not depend on the presence or absence of the corpus luteum, on the side, size and weight of the ovaries or on the type of oocytes recovered. The proportion of good quality oocytes showing cumulus expansion and extrusion of the first polar body were 79.3% and 46.1% when cultured with gonadotropins. In vitro maturation studies indicated that the proportion of oocytes that reached MII stage was significantly higher when good quality oocytes (68%) were used compared with fair quality oocytes (48%) when cultured in the presence of gonadotropins. Furthermore, fifty eight percent of the in vitro matured oocytes cleaved, and thirteen percent of the cleaved oocytes developed into blastocysts. These findings suggest that the oocytes recovered from postmortem ovaries of the blackbuck can be utilized for production of embryos.

Mature porcine oocytes containing first polar bodies (Pb I) were obtained byin vitroculture of follicle oocytes from ovaries obtained from a local abattoir, and zygotes with second polar bodies (Pb II) were grown afterin vitrofertilization of the mature oocytes. Extrusion, biological activity and morphology of Pb I and Pb II were statistically analysed. Polar bodies were isolated and collected from oocytes by enzyme digestion or micromanipulation. Their vigour under different preservation conditions was analysed and evaluated using a Trypan blue staining method. The results showed that 66.7% of the oocytes extruded Pb I after 40 h ofin vitromature culture of oocytes, and 49.7% of the zygotes extruded Pb II 20 h afterin vitrofertilization. The efficiency of isolation of Pb II by micromanipulation significantly exceeded that by enzyme digestion, the Pb I and Pb II isolated by micromanipulation presenting with good vigour and normal morphology (95.3% versus 58.9%). The survival rates of Pb I and Pb II were 63.3% and 93.1% for 4 h at 39°C, 85.0% and 72.9% for 40 h at 4°C, and over 95.0% and 84.6% for less than 7 days at −20°C. In comparison with the above preservation conditions for Pb I and Pb II, the results for cryopreservation were best, with rates of survival as high as 89.1% for Pb I and 87.9% for Pb II for preservation periods of over a month, and rates of normal morphology of 97.8% and 95.7%, respectively. The Pb I and Pb II could be isolated and preserved effectively, for use in further research on the recombination of oocytes and zygotes.

Cumulus cells (CCs) are of great importance in oocyte development and maturation in many species, but detailed influence of CCs has not been extensively examined, especially on rabbit. The present study was designed to investigate the effects of CCs and the elongation of in vitro maturation (IVM) time on rabbit oocyte nuclear and ooplasmic maturation and survival. Cumulus oocyte complexes (COCs) and naked oocytes (NOs) were recovered directly from rabbits super-ovulated with eCG. Corona-enclosed oocytes (COs) and denuded oocytes (DOs) were obtained from COCs after removing a part or whole of CCs. The oocytes were cultured in the following seven groups. (i) Cumulus cell enclosed oocytes (CEOs) were cultured alone (CEOs); (ii) COs were cultured alone (COs); (iii) DOs were cultured alone (DOs); (iv) NOs were cultured alone; (v) DOs were co-cultured with COCs [DOs(COCs)]; (vi) DOs were co-cultured with CCs [DOs(CCs)]; (vii) NOs were co-cultured with CCs [NOs(CCs)]. After the oocytes were cultured for 24 and 30 h, the nuclear maturation was evaluated by first polar body (PB1) extrusion while the ooplasmic maturation was evaluated by the cleavage rate after parthenogenetic activation. The results showed that the nuclear maturation rate of CEOs, COs, DOs(COCs) and DOs(CCs) after 24 h incubation were significantly different from each other (por = 0.05), the rate of DOs(CCs) was similar to that of DOs (por = 0.05). The cleavage rates in the first two groups were significantly higher than those of the others (p0.05). For oocytes cultured for 30 h, the nuclear maturation rates were significantly different for each culture model (p0.05). The cleavage rates in first two groups were significantly higher than those of others (p0.05). Both the nuclear and cleavage rates significantly increased when the culture time of DOs(COCs) was prolonged from 24 to 30 h. DOs(CCs) nuclear maturation was significantly improved when the culture time was prolonged from 24 to 30 h, but the ooplasmic maturation was not. Few NOs incubated with or without CCs accomplished nuclear maturation (approximately 2% both), even when the culture time was prolonged from 24 to 30 h. The oocyte degeneration rates were significantly different for each culture model after both 24 and 30 h incubation (por = 0.05). There was no significant difference in oocyte degeneration in the same groups between 24 and 30 h incubation (p0.05). The results suggest that rabbit CCs affect oocyte nuclear and ooplasmic maturation, and their survival. The prolongation of the culture time of rabbit oocyte from 24 to 30 h improves the nuclear and ooplasmic maturation differently in the present system. Rabbit oocytes free of CCs, especially NOs, show weak meiotic resumption potential and compromised viability, which cannot be improved by co-culture with dispersed CCs. The degeneration mostly happens at early time of IVM.

The location of an oocyte spindle can be identified by observing metaphase II (M II) oocytes with a LC-Polscope prior to performing intracytoplasmatic sperm injection (ICSI). Spindle damage caused by ICSI procedure raises concerns as the spindle is not always adjacent to the first polar body (PB1) and is also seen in areas penetrated by ICSI needles. LC-Polscope ICSI guided by spindle location demonstrated a significantly lower abnormal fertilization rate compared to conventional ICSI guided by location of PB1. While spindles visualized by LC-Polscope accounted for 80% of oocytes, differences in fertilization rate, good cleavage rate and blastocyst development rate were observed between visible and invisible spindle groups. These results indicate the possibility of using spindle visibility as an indicator for determining oocyte quality. It also suggests a possible correlation between the retardance of spindle image on LC-Polscope and the blastocyst development rate.

The aim of this study was to evaluate the stability of the aneuploidy rate of the first polar body. Knowing the stability of the oocyte aneuploidy rate for each patient would allow the first analysis to be used as a prognostic tool for further attempts at intracytoplasmic sperm injection (ICSI). After a first unsuccessful ICSI attempt with preconceptional screening, 24 women underwent a second attempt. First polar body aneuploidy rates were compared in the course of two successive ovarian stimulations. The first polar body was biopsied after laser dissection of the zona pellucida and five chromosomes were analysed using the MultiVysion™ polar body multicolour probe panel. A total of 200 polar bodies were analysed; 91 and 109 in the first and second ICSI attempts, respectively. The total aneuploidy rate was identical in the first and second attempts; 44.0% (40/91) and 44.0% (48/109), respectively. The first evaluation of the aneuploidy rate was statistically (P = 0.0007) correlated with the second, with a correlation coefficient, r = 0.707. The stability of the aneuploidy rate in different cohorts from the same patient, if confirmed in a larger series, makes this parameter a useful tool for counselling couples.

The aim of this work was to improve the rate of conventionally blind enucleation for bovine somatic cell nuclear transfer. The cross section of a 0.5 ml Eppendorf tube was attached with a sheet of 400 mesh/inch(2)-cell screen after the bottom of the Eppendorf tube had been cut, and put into a 1 mL Eppendorf tube. In experiment 1, the oocytes in the metaphase II stage were placed on the membrane in the Eppendorf tube, and centrifuged at 1,000, 2,000, or 3,000 r/min for 10 min, respectively. The oocytes were stained with Hoechst 33342 and then the relative position of the first polar body to the chromosomes, and the efficiency of enucleation were evaluated. In experiment 2, enucleated oocytes were fused with granulosa cells, following centrifugation and enucleation, and the potential development of the reconstituted embryos was estimated. The results indicated that the rate of enucleation in oocytes after centrifugation at 2,000 r/min for 10 min was 86.6% with an angle less then 20 degrees between the first polar body and chromosomes. The rate of enucleation in cells spun at 2,000 r/min was higher than that of controls (87.4% vs. 64.4%, P0.05). Furthermore, centrifugation of recipient oocytes did not have a detrimental effect on the development of reconstituted embryos following nuclear transfer. In conclusion, centrifugation assisted enucleation may significantly improve the rate of bovine oocyte enucleation.

Induction to tetraploidy by inhibition of the first polar body and first cellular division in fertilized eggs from diploids produced an occasional small percentage of tetraploid D-larvae, but was not successful in multiple experiments with individual or combined spawns in producing juvenile or adult tetraploids. However, when inhibition of the first polar body in eggs from triploids was done, a larger percentage of tetraploids was evidenced by flow cytometry of D-larvae, and viable larvae were obtained in one out of six induced batches. Six individuals survived to pre-adult size, five being tetraploids and one a mosaic (diploid-tetraploid). Based on these results, further efforts in producing tetraploid catarina scallop should be centered on the method involving inhibition of the first polar body in eggs from triploids, fertilized with haploid sperm.

Abnormal oocyte spindle is frequently associated with the infertility of aged women. Directly manipulating the metaphase I (MI) spindle may be a feasible method to overcome this kind of problem. Here, we report that the MI meiotic spindle can be removed from MI mouse oocytes and will autonomously divide into two daughter cells with the same size, morphology and an equal number of chromosomes after culture for 5 h in maturation medium. The division rate of the MI spindle reached 56% after 10-15 h of culture. After transferring the MI meiotic spindle into synchronous ooplasm by electrofusion, about 61% of the reconstructed oocytes continued to complete the first meiosis and extruded a normal first polar body. The matured reconstructed oocytes can also be fertilised. Approximately 50% of the 2-cell embryos developed to the morula stage after in vitro culture.

In vitro maturation is the crucial step for in vitro embryo production. It needs a large number of oocytes as source gamet cells recovered. The present study is aimed to assess the influence of corpus luteum on the average number oocytes harvested, COCs quality and subsequent maturation of immature oocytes recovered from sheep ovaries. Sheep ovaries were collected from local slaughterhouse and COCs were collected by using slicing method. Collected COCs were graded into three categories dependent upon cumulus cells surrounding them and the homogenous of cytoplasm. COCs were maturated in maturation media at 5% CO2 for 24 hours. Maturation of oocytes evaluated base on the expansion of cumulus cells and extrusion of the first polar body. There was significantly higher on average of COCs harvested from ovaries with corpus luteum compared without corpus luteum. The presence of Corpus luteum did not affect the COCs quality and ability to reach the maturation stage. However, there was a dramatic effect of cultured COCs quality on maturation rate both groups. Collectively, these results indicate that COCs quality is the main factor affecting the subsequent of oocytes matured in vitro. Keywords: Corpus luteum; cumulus oocyte complex; in vitro maturation; maturation rate; ovaries

This study was conducted to examine if oocytes obtained from one day preserved equine ovaries could be used for IVM and IVF research. Ovaries were transported at 20°C over a long distance from a slaughterhouse to a laboratory taking about 24 h. Oocytes with compact cumulus cell layers were collected from ovaries and cultured for 42 h in IVMD101 medium at 38.5°C in 5% CO2 in air. The proportions of metaphase-II stage oocytes increased significantly between 24 and 30 h of culture and reached a plateau at 30 h. After 30 h of culture, the denuded oocytes with a first polar body were subjected to intracytoplasmic sperm injection (ICSI) using frozen-thawed stallion spermatozoa. The ICSI oocytes were activated by 10 min treatment with ionophore A23187 and 6 h treatment with cycloheximide, and cultured for up to 10 days in CR1aa medium. The proportion of ICSI oocytes fertilized normally (defined as those with 2 pronuclei and 2 polar bodies) was 44%. In vitro culture of ICSI oocytes resulted in a cleavage rate of 52% and a blastocyst development rate of 9%. These results indicate that equine oocytes derived from one day preserved ovaries can reach metaphase-II stage during 30 h of IVM, and that IVM oocytes can develop into blastocysts after ICSI.

In vitro matured bovine and porcine oocytes were enucleated by aspiration of the first polar body and adjacent cytoplasm after removing the cumulus cells by vortexing and the factors affecting enucleation rate were investigated. The results indicate that vortexing cumulus-oocyte complexes for long periods in hyperosmotic media moves the first polar body and reduces the enucleation rate. Removal of 20% cytoplasmic volume increased enucleation rate compared with that of 10% and showed no deleterious effect on subsequent developmental capacity of reconstructed bovine embryos. Aging of oocytes could also reduce enucleation efficiency by inducing the migration of the chromatin material to the center of the oocyte. Therefore, we recommend to enucleate newly matured bovine and porcine oocytes by removing the first polar body and adjacent 20% of ooplasm after the removal of cumulus cells by vortexing for a short period using a hypo-osmotic media to achieve a high enucleation rate .