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PubMed1988

Fertilization of human oocytes by sperm from infertile males after zona pellucida drilling.

Infertile couples who had failed to achieve fertilization of oocytes in previous trials of in vitro fertilization (IVF) were treated by IVF with zona pellucida drilling. Zona drilling entails use of micromanipulation to introduce a gap in the zona pellucida either mechanically or by localized application of a zona solvent from a microneedle. Ten couples were treated, from whom 63 oocytes were recovered for manipulation. Sixteen eggs were denuded of the cumulus oophorus only, and the remaining 47 eggs were subjected to zona drilling. Of the 16 eggs denuded but not drilled, 4 (25%) were fertilized. Of the 47 oocytes drilled, 31 survived (67%) and 10 of the surviving eggs (32%) were fertilized. The polyspermy rate for drilled eggs that fertilized was high (5/10, 50%), and polyspermic eggs were often penetrated by more than two spermatozoa. The remaining five eggs fertilized after drilling were diploid fertilizations, and in three cases cleavage was followed by embryo transfer, although pregnancies were not obtained. These data indicate that zona drilling has the potential for establishing pregnancies in instances where treatment by standard IVF would fail. In addition, results indicate that the block to polyspermy in human eggs occurs at the level of the zona pellucida.

Female↗
PubMed1989

A zona opening procedure for improving in vitro fertilization at low sperm concentrations: a mouse model.

Micromanipulation was used for creating a small opening in the zona pellucida without damaging the ooplasm of oocytes. This was applied in mouse in vitro fertilization as a model for inseminating oocytes with low sperm concentrations. It was found that the opening in the zona did not influence the fertilization rate at 10(5) sperm/ml, but when the sperm concentration was decreased to 10(4)/ml, the oocytes containing the break had a significantly higher fertilization rate and blastocyst development, compared with their intact controls. The zona cracking procedure did not increase parthenogenetic activation nor polyspermic fertilization, although it did promote early hatching of some blastocysts. The birthrate of normal offspring was not affected by exposing oocytes to the zona opening process.

Animals↗
PubMed1998

Profiling assisted reproductive technology: outcomes and quality of infertility management.

OBJECTIVE: To critically appraise the content of the American Society for Reproductive Medicine (ASRM)/Society for Reproductive Technology (SART) Registry. DESIGN: English-language literature review. PATIENT(S): Women undergoing treatment with assisted reproductive technology (ART). INTERVENTION(S): Current ART treatments, including IVF, GIFT, zygote intrafollopian transfer (ZIFT), oocyte micromanipulation, and cryopreserved embryo transfers. MAIN OUTCOME MEASURE(S): Compliance with clinical practice guidelines, and casemix-adjusted rates of live delivery, clinical pregnancy, ectopic pregnancy, miscarriage, birth defects, implantation, fertilization, and retrieval. RESULT(S): Outcomes should be adjusted for variation in patient characteristics known to affect prognosis, including maternal age, the duration of infertility, the presumed cause(s) of infertility, the patient's prior history of treatment for infertility, and diethylstilbestrol exposure. Outcome rates should be reported using the patient as the denominator, as well as cycle, retrieval, and transfer. The statistical significance of observed differences in events rates should be indicated. Because widely accepted clinical practice guidelines related to performance of ART procedures are not available, compliance with practice guidelines cannot currently be assessed. CONCLUSION(S): Reports based on ASRM/SART Registry data can be enhanced by refined casemix adjustment, assessing outcome rates per patient, as well as per component of ART procedure, and by providing an indication of the statistical significance of observed differences in event rates. In addition, a critical appraisal of available evidence related to particular aspects of infertility management would help clarify the areas in which there is an evidentiary basis for formulation of practice guidelines, as well as topics requiring additional clinical research.

Cryopreservation↗
PubMed1998

Assisted reproductive technology in the United States and Canada: 1995 results generated from the American Society for Reproductive Medicine/Society for Assisted Reproductive Technology Registry.

OBJECTIVE: To summarize the procedures and outcomes of assisted reproductive technology (ART) initiated in the United States and Canada in 1995. DESIGN: Data were collected in the Society for Assisted Reproductive Technology database program and cycle reporting forms and were submitted to the American Society for Reproductive Medicine/Society for Assisted Reproductive Technology Registry. PARTICIPANT(S): Two hundred eighty-one programs submitted data on procedures performed in 1995. Data were collected after November 1996 so that outcome of all pregnancies established would be known. MAIN OUTCOME MEASURE(S): Procedural outcomes measured included clinical pregnancy, ectopic pregnancy, abortion, stillbirth, delivery, and congenital abnormality. RESULT(S): Programs reported initiating 59,142 cycles of ART treatment, including frozen embryo and donor oocyte cycles. Of these, 41,087 cycles initiated were IVF (with and without micromanipulation) with 22.5% deliveries per retrieval; 3,741 were cycles of gamete intrafallopian transfer with 27.0% deliveries per retrieval; 1,078 were cycles of zygote intrafallopian transfer with 27.9% deliveries per retrieval. In addition to these cycles initiated in 1995, 8,453 frozen embryo thaw procedures were reported, either as separate procedures or in combination with other ART procedures with 15.2% deliveries per transfer, 3,555 donor oocyte cycles were initiated with an overall success of 36.0% deliveries per transfer, 1,028 cryopreserved embryo thaw procedures from donated oocyte procedures with an overall success of 16.8% deliveries per transfer, and 200 ART treatment cycles in which a host uterus was used were initiated with an overall success of 34.9% deliveries per ET. As a result of all procedures, a total of 11,631 deliveries were reported, resulting in 16,520 neonates. CONCLUSION(S): In 1995, there were more programs reporting ART treatment and a significant (19.3%) increase in reported cycles. In comparable cycle types, overall average success rates (deliveries per transfer) exhibited a 0.6% increase or a 2.5% increase over the rates in the 1994 reported summaries.

Adult↗
PubMed1999

Assisted reproductive technology in the United States: 1996 results generated from the American Society for Reproductive Medicine/Society for Assisted Reproductive Technology Registry.

OBJECTIVE: To summarize the procedures and outcomes of assisted reproductive technology (ART) initiated in the United States in 1996. DESIGN: Data were collected electronically using the Society for Assisted Reproductive Technology Clinical Outcome Reporting System software and submitted to the American Society for Reproductive Medicine/Society for Assisted Reproductive Technology Registry. PARTICIPANT(S): Three hundred programs submitted data on procedures performed in 1996. Data were collected after November 1997 so that the outcome of all pregnancies established would be known. MAIN OUTCOME MEASURE(S): Procedural outcomes measured included clinical pregnancy, ectopic pregnancy, abortion, stillbirth, delivery, and congenital abnormality. RESULT(S): Programs reported initiating 65,863 cycles of ART treatment, including frozen embryo and donor oocyte cycles. Of these. 44,647 cycles initiated were in vitro fertilization (IVF) (with and without micromanipulation) with 26.0% deliveries per retrieval; 2,879 were cycles of gamete intrafallopian transfer (GIFT) with 29.0% deliveries per retrieval; 1,200 were cycles of zygote intrafallopian transfer (ZIFT) with 30.9% deliveries per retrieval. In addition to these cycles initiated in 1996, 9,610 frozen embryo transfer procedures were initiated with 16.8% deliveries per transfer, 3,768 donor oocyte cycles were initiated with an overall success of 39.1% deliveries per transfer, 1,096 cryopreserved embryo transfers from donated oocytes procedures with an overall success of 20.8% deliveries per transfer, and 688 ART treatment cycles using a host uterus were initiated with an overall success of 31.3% deliveries per embryo transfer. Also, 1,341 cycles were reported as combinations of more than one treatment type, 19 cycles as research, 311 as embryo banking, and 304 as other (unclassified) cycle types. As a result of all procedures, a total of 14,702 deliveries were reported resulting in 21,196 neonates. CONCLUSION(S): In 1996, there were more programs reporting ART treatment and a significant (11.3%) increase in reported cycles. In comparable cycle types, overall average success rates (deliveries per retrieval) exhibited an actual increase of 3.5% (this is an increase of 15.8% when compared to the success rate for 1995).

Female↗
PubMedDate not supplied

Design of computer-generated hologram with ring focus for nonmechanical corneal trephination with Er:YAG laser in penetrating keratoplasty.

PURPOSE: To calculate a beam-shaping optical element for homogeneous intensity distribution within a focal ring to be used in nonmechanical trephination with the Er:YAG laser in penetrating keratoplasty instead of a spot guiding device. METHODS: The phase distribution behind a holographic optical element (HOE) k psi(u) can be described by the addition of the hologram phase phiH(u) to the beam phase phiE(u): k psi(u) = phiH(u) + phiE(u), k = 2pi/lambda, where u denotes the coordinates inside the hologram aperture, k an integer, and lambda the laser wavelength. To avoid discontinuous wavefronts leading to speckle noise, a smooth phase function is necessary. After transforming the hologram aperture coordinates into the focal plane x in a focal distance f, psi can be retrieved from the slope equation: inverted delta psi(u) = x(u) - u/f. RESULTS: Creating a ring focus can be reduced to an essentially one-dimensional problem by separation of variables due to the symmetry condition. We calculated a computer-generated eight-level phase-only HOE with 4096 x 4096 pixels from a Gaussian-distributed 2.94 Er:YAG laser spot with a beam diameter of 10 mm and a focal distance of 100 mm. Thereby, a ring focus with an inner/outer radius of 7/8 mm can be created. To avoid Poisson's spo, the symmetry of the problem was broken by circular modulation of the phase leading to a spiral-like structure. The calculated efficiency of the HOE relating the energy within the ring to the total energy was 91%. CONCLUSION: With an HOE it is possible to redistribute the energy along the desired focal ring. The HOE design can be adapted to the intensity distribution of the impinging laser beam with its characteristic aperture shape. A circular homogeneous corneal trephination depth is possible, because the energy fluctuation from pulse to pulse does not locally affect the ablation process. A ring focus for the Er:YAG laser has the potential to render superfluous a manual beam control via micromanipulator and to allow a more rapid and more regular corneal trephination along aperture masks.

Cornea↗
PubMed2002

pH Heterogeneity of human and rabbit atherosclerotic plaques; a new insight into detection of vulnerable plaque.

BACKGROUND: Atherosclerotic plaques are heterogeneous with respect to inflammation, calcification, vascularity, oxygen, and temperature. We hypothesized that they also vary in pH and measured pH in living human carotid endarterectomized atherosclerotic plaques (CEA), Watanabe heritable hyperlipidemic (WHHL) rabbit aortas and human umbilical arteries (HUA). METHODS AND RESULTS: We measured pH of CEA of 48 patients, nine WHHL rabbit aortas and 11 HUA specimens (as controls) using a glass type microelectrode mounted on a micromanipulator in a 37 degrees C incubator. We also used single emission and also dual emission fluorescence ratio imaging microscopy employing pH-sensitive probes to confirm pH heterogeneity. Mean pH measured at 415 points of CEA was 7.55+/-0.32; at 275 points of WHHL rabbit aortas it was 7.40+/-0.43; and in 233 points of HUA it was 7.24+/-0.1. In CEA, pH of yellow (lipid-rich) areas was significantly lower than pH in calcified areas (7.15+/-0.01 vs. 7.73+/-0.01, P<0.0001). The coefficients of variation (heterogeneity) of pH in CEA, WHHL rabbit aortas, and HUA were 0.038+/-0.010, 0.039+/-0.007, and 0.009+/-0.003, respectively (P=0.0001). Fluorescence microscopic imaging confirmed pH heterogeneity in both humans and rabbits but not in HUA. In a variance components analysis 82% of the heterogeneity was due to the within-plaque variation and 2% was attributable to between-plaque variation. CONCLUSIONS: Our findings support the hypothesis of pH heterogeneity in plaques, and suggest a possible role for detecting low pH in the detection of plaque vulnerability. The source of pH heterogeneity particularly acidic pH, its impact on the stability of plaques and its potential clinical utility in locating vulnerable plaques remain to be evaluated.

Animals↗
PubMed2000

A newly designed tensile tester for cells and its application to fibroblasts.

A tensile test system for cells has been designed and applied to fibroblasts from the rabbit patellar tendon. It consists of a thermostatic test chamber, an inverted fluorescence microscope, micromanipulators, a direct drive linear actuator, a cantilever-type load cell, and a video dimension analyzer (VDA). The test chamber and the microscope are mounted on a vibration isolator. A cell floated in Hanks' balanced salt solution of 37 degrees C is gripped with a pair of micropipettes which have very fine tips (outer diameter = 20 approximately 30 microm, inner diameter = 3 approximately 5 microm) and are coated with a cell adhesive, Cell-Tak, at their ends. One of the micropipettes is fixed to the load cell; the other one is attached to the linear actuator which is used to stretch the cell. Load applied to the cell is measured with the load cell, while elongation of the cell is determined with the VDA using the images of the ends of the micropipettes as markers. The measurement accuracy of the load cell was +/-0.05 microN. All the fibroblasts tested were firmly attached to the micropipettes during tensile testing, and showed local non-uniform deformation. The maximum load and elongation to failure of the cells were 0.9+/-0.2 microN and 86+/-24 microm, respectively.

Animals↗
PubMed2002

Determination of purine and pyrimidine bases in DNA by micellar electrokinetic capillary chromatography with electrochemical detection.

A method based on micellar electrokinetic capillary chromatography with electrochemical detection was developed for the determination of cytosine, 5-methylcytosine (5-MC), thymine, adenine, and guanine in the hydrolysates of DNA. The working electrode was fabricated in a novel self-positioning carbon disc electrode system that can align the capillary outlet with the working electrode without a three-dimensional micromanipulator. The five analytes could be well separated within 10 min in a 40 cm length capillary at a separation voltage of 9 kV in a 40 mmol/l borate buffer (pH 10.0) containing 100 mmol/l sodium dodecyl sulfate. Good linearity was observed between peak current and concentration of bases over three orders of magnitude with the detection limits (SIN=3) ranging from 1.28 x 10(-6) to 5.02 x 10(-6) mol/l. This proposed method demonstrated long-term stability and reproducibility with relative standard deviations of less than 5% for both migration time and peak current (n=7). It has been successfully applied to determine bases including 5-MC in the hydrolysates of fish sperm DNA, calf thymus DNA, and DNA isolated from spleen cells of female mice.

Chromatography, Micellar Electrokinetic Capillary↗
PubMed2003

Direct measurement of interaction forces between a single bacterium and a flat plate.

A technique for precisely measuring the equilibrium and viscous interaction forces between a single bacterium and a flat surface as functions of separation distance is described. A single-beam gradient optical trap was used to micromanipulate the bacterium against a flat surface while evanescent wave light scattering was used to measure separation distances. Calibrating the optical trap far from the surface allowed the trapped bacterium to be used as a force probe. Equilibrium force-distance profiles were determined by measuring the deflection of the cell from the center of the optical trap at various trap positions. Simultaneously, viscous forces were determined by measuring the relaxation time for the fluctuating bacterium. Absolute distances were determined using a best-fit approximation to the theoretical prediction for the hindered mobility of a diffusing sphere near a wall. Using this approach, forces in the range from 0.01 to 4 pN were measured at near-nanometer resolution between Staphylococcus aureus and glass that was bare or coated with adsorbed protein.

Adsorption↗
PubMed2003

Attachment forces of the hemelytra-locking mechanisms in aquatic bugs (Heteroptera: Belostomatidae).

Combined hemelytra-locking system of Heteroptera, consisting of several locking mechanisms, aids the mechanical stabilisation of the body at rest, resists external loads, and keeps air stored with the option to easily unlock hemelytra prior to flight. The resistance to unlocking of the hemelytron was measured (in mN) with the aid of a load cell force transducer combined with a three-axial micromanipulator. It is shown that macro- and microstructural features of several submechanisms are responsible for their directionality. The highest resistance to unlocking was measured in lateral and dorsal directions. Summarised force of separately measured submechanisms was considerably lower than the force measured in the combined mechanism. Each submechanism is optimised for achieving high resistance to the hemelytron uncoupling in particular direction(s) and to be easily unlocked in another direction. It was demonstrated in the high-speed videorecordings that hemelytra uncoupling is promoted by their short anterior displacement.

Animals↗
PubMed2003

Robotics and neurosurgery.

Ultimately, neurosurgery performed via a robotic interface will serve to improve the standard of a neurosurgeon's skills, thus making a good surgeon a better surgeon. In fact, computer and robotic instrumentation will become allies to the neurosurgeon through the use of these technologies in training, diagnostic, and surgical events. Nonetheless, these technologies are still in an early stage of development, and each device developed will entail its own set of challenges and limitations for use in clinical settings. The future operating room should be regarded as an integrated information system incorporating robotic surgical navigators and telecontrolled micromanipulators, with the capabilities of all principal neurosurgical concepts, sharing information, and under the control of a single person, the neurosurgeon. The eventual integration of robotic technology into mainstream clinical neurosurgery offers the promise of a future of safer, more accurate, and less invasive surgery that will result in improved patient outcome.

Clinical Competence↗
PubMed1988

Retinal transplants and optic nerve bridges: possible strategies for visual recovery as a result of trauma or disease.

From the review of the current literature it is quite evident that some exciting prospects are on the horizon which will help to better explain the development and functioning of the visual system. In addition, the new technology of CNS tissue grafting coupled to other newly emerging technologies (i.e., microsurgical, microinjection, and micromanipulative techniques coupled with our knowledge of immunosuppressive methods) will allow for a realistic approach in exploring possible strategies for visual recovery as a result of trauma or disease within the near future. One specific area of research that hopefully will emerge from this new body of knowledge comes from the realization that at the present time there is no effective therapy for practically all types of hereditary retinal degenerative disorders in man. It would seem most appropriate to take advantage of the new neuronal transplantation technology mentioned in this article and the availability of hereditary retinal degeneration models in the hope of developing new methods for a therapeutic approach to this problem. Such an approach could involve replacing the abnormal, absent, and/or lost host retinal cells with tissue from healthy donors by means of a grafting technique with the goal of arresting and/or reversing the disease process. Of course, this is but one example of the many challenges in this area of research which increasingly appear to be within our grasp.

Animals↗
PubMed2002

Flexible polyacrylamide substrata for the analysis of mechanical interactions at cell-substratum adhesions.

We have described a powerful tool for the study of mechanical interactions between cells and their physical environment. Although the approach has already been used in a variety of ways to measure traction forces and to characterize active and passive responses of cultured cells to mechanical stimulation, it can be extended easily and combined with other microscopic approaches, including fluorescent analog imaging (Beningo et al., 2001), photobleaching, calcium imaging, micromanipulation, and electrophysiology. This method will be particularly useful for studying the functions of various components at focal adhesions, and the effects of mechanical forces on focal adhesion-mediated signal transduction. In addition, the method can be extended to a 3D setting, e.g., by sandwiching cultured cells between two layers of polyacrylamide to create an environment mimicking that in the tissue of a multicellular organism. Whereas chemical interactions between cells and the environment have been investigated extensively, many important questions remain as to the role of physical forces in cellular functions and the interplay between chemical and physical mechanisms of communication. The present approach, as well as other approaches capable of probing physical interactions, should fill in this important gap in the near future.

Acrylic Resins↗
PubMed2001

Delipidating in vitro-produced bovine zygotes: effect on further development and consequences for freezability.

To study the effect of partial removal of intracytoplasmatic lipids from bovine zygotes on their in vitro and in vivo survival, presumptive zygotes were delipidated by micromanipulation and cocultured with Vero cells in B2+10% FCS. Blastocyst rates of delipidated (n=960), sham (centrifuged but not delipidated, n=830) and control embryos (n=950) were 42.1, 42.3 and 39.9% respectively (P > 0.05). Day 7 blastocysts derived from delipidated zygotes had a mean of 123.9 +/-45.6 nuclei compared to 137.5+/-32.9 for control blastocysts (P > 0.05). The full-term development of delipidated blastocysts after single transfer to recipients was similar to that of control IVF blastocysts (41.2% vs 45.4% respectively). To assess the effect of delipidation on the embryo tolerance to freezing/thawing, delipidated (n=73), control (n=67) and sham (n=50) Day 7 blastocysts were frozen in 1.36 M glycerol + 0.25 M sucrose in PBS. After thawing, embryos were cocultured for 72 h with Vero cells in B2+10% FCS. Survival rates at 24 h were not significantly different between groups. However, in the delipidated group, the survival rate after 48 h in culture was significantly higher than in the control group (56.2 vs 39.8, P < 0.02), resulting in a higher hatching rate after 3 days in culture (45.2 vs 22.4, P < 0.02). Pregnancy rates for delipidated and control frozen/thawed embryos were respectively 10.5 and 22.2% (P > 0.05). Electron microscopic observations showed much fewer lipid droplets (and smaller) in delipated blastocysts than in controls. Taken together, our data show that delipidation of one cell stage bovine embryos is compatible with their normal development to term and has a beneficial effect on their tolerance to freezing and thawing at the blastocyst stage. This procedure, however, alters the developmental potential of such blastocysts, suggesting that maternally inherited lipid stores interfere with metabolic recovery after thawing.

Animals↗
PubMed2001

Progress in reproductive biotechnology in swine.

This article summarizes recent progress in reproductive biotechnology in swine with special reference to in vitro production of embryos, generation of identical multiples, and transgenic pigs useful for xenotransplantation. In vitro production (in vitro maturation, in vitro fertilization, and in vitro culture) of viable porcine embryos is possible, although with much lower success rates than in cattle. The main problems are insufficient cytoplasmic maturation of porcine oocytes, a high proportion of polyspermic fertilization and a low proportion of blastocysts that, in addition, are characterized by a low number of cells, hampering their development in vivo upon transfer to recipients. Microsurgical bisection of morula and blastocyst stage embryos leads to a 2 to 3% monozygotic twinning rate of the transferred demiembryos, which is similar to that in rabbits and mice but considerably lower than in ruminants. It was found that with decreasing quality an increasing proportion of demi-embryos did not possess an inner cell mass. Porcine individual blastomeres derived from 4- and 8-cell embryos can be cultured in defined medium to the blastocyst stage. Leukemia inhibitory factor has been shown to be effective at defined embryonic stages and supports the formation of the inner cell mass in cultured isolated blastomeres in a concentration-dependent manner. For maintaining pregnancies with micromanipulated porcine embryos, it is not necessary to transfer extraordinarily high numbers of embryos. Porcine nuclear transfer is still struggling from the inefficiency of producing normally functioning blastocysts. Blastomeres, blastocyst-derived cells, fibroblasts and granulosa cells have been employed as donor cells in porcine nuclear transfer and have yielded blastocysts. Recently, the generation of the first piglets from somatic cell nuclear transfer has been achieved. DNA-microinjection into pronuclei of porcine zygotes has reliably resulted in the generation of transgenic pigs, which have special importance for the production of valuable pharmaceutical proteins in milk and xenotransplantation. It has been demonstrated that by expression of human complement regulatory proteins in transgenic pigs the hyperacute rejection response occurring after xenotransplantation can be overcome in a clinically relevant manner. Although biotechnological procedures in swine have recently undergone tremendous progress, the development is still lagging behind that in cattle and sheep. With regard to genetic engineering, considerable progress will originate from the possibility of employing homologous recombination in somatic cell lines and their subsequent use in nuclear transfer. In combination with the increasing knowledge in gene sequences this will allow in the foreseeable future widespread use in the pig industry either for agricultural or biomedical purposes.

Animals↗
PubMed2002

Advancements in cryopreservation of domestic animal embryos.

The development of embryo freezing technologies revolutionized cattle breeding. Since then, advancements in cryobiology, cell biology, and domestic animal embryology have enabled the development of embryo preservation methodologies for our other domestic animal species, including sheep and goats. Recently, technologies have been developed to cryopreserve pig embryos, notorious for their extreme sensitivity to cooling; horse embryo cryopreservation is in its infancy. While cryopreservation can enhance the utilization of in vitro embryo production technologies, cryosurvival of in vitro-produced (IVP) or micromanipulated embryos is less than that of in vivo-derived embryos. This review outlines recent efforts in livestock embryo cryopreservation. In the near future, use of preserved embryos could be a routine breeding alternative for all livestock producers providing 1) preservation methods for maternal germplasm, 2) global genetic transport, 3) increased selection pressure within herds, 4) breeding line regeneration or proliferation, and 5) methodology for genetic rescue.

Animals↗
PubMed2002

Effect of co-culture with theca interna on nuclear maturation of horse oocytes with low meiotic competence, and subsequent fusion and activation rates after nuclear transfer.

We conducted this study to examine whether or not co-culture with theca cells improves the maturation rate of horse oocytes with compact cumuli and to evaluate the cytoplasmic competence of oocytes after maturation by assessing fusion, activation and cleavage rates after nuclear transfer. We collected oocytes by scraping follicles from slaughterhouse-derived ovaries and classified them as having an expanded or a compact cumulus. Expanded oocytes were matured in M199 supplemented with 10% FBS and 5 microU/ml FSH for 24 h: compact oocytes were cultured in the same medium, or they were co-cultured in the same medium with theca interna explants, for 24 or 42 h. Oocytes were held with or without 10 microg/ml cytochalasin B, before washing and micromanipulation. and they were fused with donor fibroblasts by electrical pulse. Fused oocytes were activated with Ca ionophore/cycloheximide, cultured for 5 days, and stained with Hoechst to assess nuclear development. We considered oocytes with an enlarged nucleus, or having cleavage with multiple nuclei, to be activated. There was no significant difference in overall maturation rate between compact oocytes cultured with theca and compact controls. When these two groups were combined, there was a significant increase in the proportion of oocytes in MII between 24 and 42 h (P < 0.05). Expanded oocytes had a significantly higher rate of maturation than did compact oocytes (64% versus 25-30%; P < 0.001). There were no significant differences in rates of successful enucleation, fusion, activation or cleavage between compact control and compact + theca oocytes, nor between compact and expanded oocytes; however, expanded oocytes treated with cytochalasin B had a significantly higher survival rate after enucleation than did untreated expanded oocytes (P < 0.05). Three embryos developed from recombined oocytes, with maximum cleavage to 10 cells. The results of this study indicate that co-culture with theca cells does not increase either nuclear or cytoplasmic maturation of compact oocytes. Cytochalasin B is helpful in increasing survival of horse oocytes during enucleation. In vitro matured equine oocytes have the potential to develop into embryos after nuclear transfer; this is the first full report of production of cloned embryos in this species.

Animals↗