In vitro technique for studying garter snake (Thamnophis sp.) development

Garter snake (Thamnophis sp.) embryos of different embryonic ages were explanted from pregnant females and grown in vitro for up to 35 days. The culture medium consisted of Eagle's Minimum Essential Medium with various organic and inorganic supplements and antibiotics. Cultured embryos were injected with tritiated thymidine, sacrificed, and processed for autoradiographic examination. The in vitro technique allowed direct visualization of external morphologic changes over time, which followed a timetable similar to that of embryos growing in vivo. This technique also allowed easier manipulation of embryos for determination of embryonic stage and for injection of tritiated thymidine. This is the first report of long-term culture of snake embryos which adds to the limited number of reptilian embryos that have been studied in vitro.     

Evaluation of developmental competence of vitrified-warmed early cleavage stage embryos and their application for chimeric mouse production.

The developmental competence of in vitro cultured embryos vitrified-warmed at an early cleavage stage (2- or 4, 8-cell stage) was examined by both direct transfer into recipient animals and after in vitro manipulation for chimeric mice production using embryonic stem (ES) cells. Vitrified-warmed embryos transferred at the morulae and blastocyst stages showed fetus development comparable to control embryos, although blastocyst development of vitrified-warmed embryos was significantly slower than that of controls. When vitrified-warmed early cleavage stage embryos were used for chimeric mouse production using ES cells, 1 to 10% of the injected or aggregated embryos developed into chimeric neonates and germ-line chimeric mice were obtained from all ES cell lines. This study indicates that embryos developed in vitro from vitrified-warmed embryos have equivalent competence with unvitrified embryos irrespective of stage of vitrification and that these vitrified-warmed embryos maintain adequate viability even after in vitro manipulation such as aggregation and microinjection with ES cells.     

Cyclic variations in incubation conditions induce adaptive responses to later heat exposure in chickens: a review

Selection programs have enabled broiler chickens to gain muscle mass without similar enlargement of the cardiovascular and respiratory systems that are essential for thermoregulatory efficiency. Meat-type chickens cope with high ambient temperature by reducing feed intake and growth during chronic and moderate heat exposure. In case of acute heat exposure, a dramatic increase in morbidity and mortality can occur. In order to alleviate heat stress in the long term, research has recently focused on early thermal manipulation. Aimed at stimulation of long-term thermotolerance, the thermal manipulation of embryos is a method based on fine tuning of incubation conditions, taking into account the level and duration of increases in temperature and relative humidity during a critical period of embryogenesis. The consequences of thermal manipulation on the performance and meat quality of broiler chickens have been explored to ensure the potential application of this strategy. The physiological basis of the method is the induction of epigenetic and metabolic mechanisms that control body temperature in the long term. Early thermal manipulation can enhance poultry resistance to environmental changes without much effect on growth performance. This review presents the main strategies of early heat exposure and the physiological concepts on which these methods were based. The cellular mechanisms potentially underlying the adaptive response are discussed as well as the potential interest of thermal manipulation of embryos for poultry production.     

Genetic manipulation of microspores and microspore-derived embryos

Summary Recent advances in plant cell and molecular biology have furthered the genetic manipulation of many plant species and advanced the options for crop improvement. Among the many targets for genetic manipulation, microspores offer several unique advantages: they are haploid, single-celled, and highly synchronized. In many plant species microspores develop into haploid embryos, and eventually haploid and doubled haploid plants, after in vitro anther or microspore culture. This induced in vitro developmental pathway of microspores, termed microspore embryogenesis, can be used to recover individual homozygous plants from microspores and microspore-derived embryos after genetic manipulation such as mutagenesis and gene transfer. The highly efficient microspore embryogenesis system inBrassica napus has been used successfully to obtain various mutants after microspore mutagenesis, and to achieve gene transfer mediated byAgrobacterium tumefaciens. Presented in the Session-in-Depth In Vitro Gametophyte Biology at the 1991 World Congress on Cell and Tissue Culture held in Anaheim, California, June 16–20, 1991.     

Using electroporation and a slot cuvette to deliver plasmid DNA to insect embryos

Microinjection is the method used almost exclusively to deliver DNA constructs to insect embryos while electroporation is commonly used for DNA delivery to bacteria, cell cultures and certain plant tissues. This communication describes a method using an easily constructed slot cuvette and the electroporation technique for transfer of DNA to insect embryos for possible use in developing methods for germline transformation. This method eliminates time-consuming individual embryo manipulation and thus far has been found to be adaptable for use on several types of insect embryos. Using this method, we show successful transfer of plasmid DNA to embryos of the corn earworm moth, Helicoverpa zea, and the house fly, Musca domestica.     

Cytochalasin B treatment of mouse oocytes during intracytoplasmic sperm injection (ICSI) increases embryo survival without impairment of development

Summary Intracytoplasmic sperm injection (ICSI) is a technique commonly used in clinical and research settings. In mouse oocytes, conventional ICSI has a poor survival rate caused by a high level of lysis. Cytochalasin B (CB) is a toxic microfilament-inhibiting agent that is known to relax the cytoskeleton and enhance the flexibility of oocytes. CB has been used widely in nuclear transfer experiments to improve the success rate of the micromanipulation, however information describing the use of CB in ICSI is limited. Here, we demonstrated that the addition of 5 μg/ml CB to the manipulation medium of ICSI procedure significantly improved the survival rate of the ICSI embryos (80.74% vs. 89.50%, p < 0.05), and that there was no harm for the in vitro or in vivo development. The birth rates and birth weights were not significantly different between the CB-treated and -untreated groups. Interestingly, the microfilaments of the ICSI embryos were almost undetectable immediately after CB treatment; however, they gradually re-appeared and had fully recovered to the normal level 2 h later. Moreover, CB did not disturb spindle rotation, second polar body formation or pronuclei migration, and had no effect on the microtubules. We thus conclude that ICSI manipulation in CB-containing medium results in significantly improved survival rate of mouse ICSI embryos, and that short-term treatment with CB during ICSI manipulation does not have adverse effects on the development of ICSI embryos.     

Isolation and use of embryonic stem cells from livestock species

Abstract

Embryonic stem (ES) cells are pluripotent cells isolated from early embryos. They proliferate in culture and retain the capacity to differentiate both in vitro and In vivo, including contributing to chimeric tissues after injection into normal blastocysts. Over the past decade ES cells have been used extensively as a model for embryogenesis. More recently they have been shown to be capable of stable integration of exogenous DNA and used for numerous studies involving genomic manipulation. ES cells provide many opportunities for genetic engineering of domestic livestock species, but to date their isolation from embryos has been documented only for the mouse and perhaps the hamster. Efforts to isolate pluripotent ES cells from embryos of domestic livestock species are described, including some of the problems encountered.     

Use of the rabbit oviduct as a screening tool for the viability of mammalian eggs

The oviducts of both oestrous and pseudopregnant rabbits can be used for the successful culture of mammalian embryos for short periods. This has alowed some selection to be made on the embryos as they are examined on at least two occasions before final transfer. Not only have pregnancy rates been normal, but in some instances they have been higher following a limited period (2–3 days) in the rabbit oviduct. It would appear that these higher pregnancy rates result from a more intensive selection of embryos at the time of transfer rather than from some substance acquired during storage in the oviduct. However, the system is not without disadvantages. There is some loss of embryos (15–30%) in the oviduct and all embryos recovered may not have developed at the normal rate.The rabbit oviduct has been used as a site of xenogenous fertilization. Initial reports indicate that success in that area is lower than when using large animals as the site of fertilization. With more widespread interest in the use of microsurgery in embryos, the rabbit oviduct has been used for the short term storage of agar cylinders and has been found to be unsuitable because of the high rate of degeneration of agar chips. However, the rabbit oviduct is still useful as an experimental tool in the manipulation of embryos from the domestic species.     

Effects of sucrose treatment on the development of mouse nuclear transfer embryos with morula blastomeres as donors

In this study, nuclear transfer (NT) embryos were produced by using C57Bl/6 mouse morula blastomeres and Kunming mouse metaphase II (MII) oocytes as donors and recipients, respectively, to investigate the effects of sucrose treatment of MII oocytes with different concentrations on the manipulation time of NT, electrofusion and the in vitro and in vivo development of reconstructed embryos. The results demonstrated that: (i) when the oocytes were enucleated with 1, 2 and 3% sucrose treatment, respectively, the enucleating rates were not affected by the different sucrose concentrations, but the manipulation time had significant difference and the mean nuclear transfer manipulation times of every oocyte were 180+/-10 s, 130+/-10 s and 120+/-10 s, respectively; (ii) different sucrose concentrations had no significant effects on the fusion rate and the in vitro developmental potential of the NT embryos (p>0.05). Furthermore, 59 embryos were transplanted into the oviducts of two recipients. In the end, three dead full-term developed fetuses were obtained on 21 days post coitus (dpc). These results suggested that the mouse MII oocytes enucleated via sucrose treatment might be an alternative source for mouse cloning and could support the embryonic NT embryos developed to term in vivo.     

The Xenopus Cell Cycle: An Overview

Oocytes, eggs and embryos from the frog Xenopus laevis have been an important model system for studying cell-cycle regulation for several decades. First, progression through meiosis in the oocyte has been extensively investigated. Oocyte maturation has been shown to involve complex networks of signal transduction pathways, culminating in the cyclic activation and inactivation of Maturation Promoting Factor (MPF), composed of cyclin B and cdc2. After fertilisation, the early embryo undergoes rapid simplified cell cycles which have been recapitulated in cell-free extracts of Xenopus eggs. Experimental manipulation of these extracts has given a wealth of biochemical information about the cell cycle, particularly concerning DNA replication and mitosis. Finally, cells of older embryos adopt a more somatic-type cell cycle and have been used to study the balance between cell cycle and differentiation during development.     

Advances on in vitro production and cryopreservation of porcine embryos

There have been intensive attempts to establish reliable in vitro production (IVP) and cryopreservation methods of embryos in pigs. Although a great deal of progress has been made, current IVP systems and cryopreservation still suffer from insufficient cytoplasmic abilities of in vitro matured oocytes, polyspermic fertilization, poor quality of in vitro produced embryos and low efficiency of embryo cryopreservation. Compared to other mammalian species, pig oocytes and embryos are characterized by large amounts of lipid content stored mainly in the form of lipid droplets in the cytoplasm. This fact has a negative influence on biotechnological applications on porcine oocytes and embryos. In this review, we will discuss recent studies about methods and techniques for modifying porcine embryo IVP system and embryo cryopreservation that produces high quality of pig blastocysts using in vitro maturation, in vitro fertilization, in vitro culture, microsurgical manipulation, addition of protein, the use of cytoskeleton stabilizing agents and various physical methods. The presented methods and techniques make it possible to modify the characteristics of oocytes and embryos and thus may become major tools in mammalian gamete and embryo agricultural or biotechnological applications in the future.     

Micromanipulation of mammalian embryos: Principles, progress and future possibilities

Numerous advances in development of techniques for manipulating mammalian embryos outside the maternal environment have been made over the past decade. Some techniques were developed primarily for use in research; others were developed in response to problems of practical livestock production but have proven useful in research as well. Embryo micromanipulation procedures are used often in conjunction with embryo transfer, and interest in these procedures was stimulated by growth of the embryo transfer industry. Included in this review are discussions of procedures for manipulation of gametes and embryos, including sperm injection into oocytes, pronuclear and nuclear transfer, embryo biopsy and splitting, experimental chimera production and isolation of embryonic stem cells.     

In vitro culture of Arabidopsis embryos within their ovules

Embryogenesis of flowering plants establishes a basic body plan with apical-basal, radial and bilateral patterns from the single-celled zygote. Arabidopsis embryogenesis exhibits a nearly invariant cell division pattern and therefore is an ideal system for studies of early plant development. However, plant embryos are difficult to access for experimental manipulation, as they develop deeply inside maternal tissues. Here we present a method for the culture of zygotic Arabidopsis embryos in vitro. The technique omits excision of the embryo by culturing the entire ovule, thus greatly facilitating the time and effort involved. It enables external manipulation of embryo development and culture from the earliest developmental stages up to maturity. Administration of various chemical treatments as well as the use of different molecular markers is demonstrated together with standard techniques for visualizing gene expression and protein localization in in vitro cultivated embryos. The presented set of techniques allows for so far unavailable molecular physiology approaches in the study of early plant development.     

The spontaneous occurrence of aortic arch and cardiac malformations in the white Leghorn chick embryo (Gallus domesticus)

Spontaneous aortic arch and cardiac malformations occur in White Leghorn chick embryos at a relatively high rate. Although this breed of Gallus domesticus is widely used for biomedical and biological research, no previous study has recorded the incidence of these defects. We found aortic arch malformations in 7.1% (14 of 196) and ventricular septal defects in 11.7% (23 of 196) of living embryos. Defects occurred alone or as a combined pattern. Our findings suggest that the cardiovascular defects in the chick embryo documented in past studies may, in some cases, have been part of normal spontaneous occurrence, rather than the major result of experimental manipulation.     

Pregnancy and live birth from nonsurgical transfer of in vivo- and in vitro-produced blastocysts in the rhesus monkey

Embryo transfer in the rhesus monkey has been historically limited to transfer of cleavage stage embryos. In order to allow genetic manipulation of rhesus embryos in vitro, without using invasive surgical techniques, it is important to explore the transfer of morula and blastocyst stage embryos. Embryos were produced by in vitro fertilization from gonadotropin-stimulated monkeys, or were obtained by nonsurgical uterine flushing of naturally mated or artificially inseminated females. Nonsurgical transfer was accomplished by inserting a metal guide through the cervix into the uterus, after which a hollow cell sampler was inserted over the guide. The guide was removed and a catheter was inserted containing one to five embryos. Several pregnancies resulted from in vitro- and in vivo-derived blastocysts, and two pregnancies were carried to term resulting in one live birth. Blood samples were collected regularly to monitor plasma levels of chorionic gonadotropin, luteinizing hormone, and progesterone. The recipients received progesterone as a subcutaneous implant or daily injections from the day of transfer. The approach described in this study provides the opportunity to explore transgenic and chimeric models in the monkey by the development of noninvasive methods to transfer late-stage embryos that have been manipulated in vitro.     

Transplantation of a polyembryonic wasp embryo: a technique for transferring endoparasitic embryo into the host egg

Concealed development of many animal embryos prevents examination of development and limits the application of embryo manipulation techniques aimed at understanding developmental processes. In embryos developing in utero, such as in mammals, it is necessary to dissect embryos from the mother and, upon manipulative intervention, to implant them back into the recipient. Parasitic wasps present a promising system for understanding the evolution of early developmental processes. In basal ectoparasitic species that lay eggs on the surface of the host, it is possible to adapt embryo manipulation techniques developed in Drosophila. However, their derived endoparasitic relatives, which exhibit various modifications of developmental programs, undergo concealed development within the host body. For example, the parasitic polyembryonic wasp Copidosoma floridanum oviposits an egg into the egg of the host moth Trichoplusia ni. The host larva emerges and the parasite undergoes development within the host body, preventing embryo manipulation as a means of examining developmental regulation. Here we present a protocol for embryo transfer that allows the transplantation of C. floridanum egg into the host egg. This approach opens a new avenue in the application of various embryo manipulation techniques aimed at understanding the evolution of embryogenesis in endoparasitic Hymenoptera. In addition, this approach has potential for the development of other tools in C. floridanum, such as transgenesis and reverse genetics, which can also be extended to other endoparasitic species.     

The analysis of mammalian development using allelic isozyme variants

The past decade has seen an explosion of interest in mammalian embryos. Techniques of molecular and genetic analysis coupled with advances in in vitro culture and experimental manipulation of mammalian embryos have provided important insights into mechanisms of embryogenesis. Many of these recent advances have been facilitated by the use of allelic isozyme variants as autonomous cell markers or representative gene products. Investigations aimed at exploring cell lineages and cell commitment, the timing and regulation of gene expression, X chromosome inactivation, and cell interactions have depended on the availability of appropriate isozyme variants. Results from such experiments are summarized here in order to demonstrate the usefulness of this approach and to stimulate its wider application in developmental biology.