The effective culture method of zona-free rabbit 1-, 2- and 4-cell embryos

The effect of modified incubation systems on the development capacity of the zona-free rabbit embryos was examined. Embryos at 1-, 2- and 4-cell stages were used. The removal of the zona pellucida was accomplished by the enzymic-mechanical technique. Denuded rabbit embryos were cultured using 3 incubation systems. In the first and the second system the embryos were cultured in microdrops. The difference between these first 2 systems concerned the volume of the microdrops and the kind of paraffin oil used. In the first system the embryos were cultured in 5mul microdrops covered with light or heavy paraffin oil; in the second system embryos were cultured in 40-mul microdrops under light paraffin oil. The third traditional system involved the incubation of embryos in glass capillaries into separated columns of medium. The percentage of blastocysts obtained from 1-cell embryos cultured in the first incubation system was 6.1% with heavy paraffin oil as the covering layer and 29.0% with light paraffin oil. In the second and third incubation systems blastocyst yield was 30.8 and 59.6%, respectively. The percentage of blastocysts obtained from 2-cell and 4-cell stage embryos with heavy paraffin oil was 18.7 and 25.0%, respectively; with light paraffin oil these figures were 40.0 and 50.0%, respectively. In the second incubation system these figures were 49.3 and 72.3%; and in the third incubation system they were 72.9 and 78.3%, respectively. The results of the experiment showed that culture into glass capillaries is undoubtedly an effecient method of culturing of the zona-free rabbit embryos.     

An alternative method for the culture of Diplostomum spathaceum (Trematoda) in chick embryos

Metacercariae of Diplostomum spathaceum were maintained in the allantoic cavities of chick embryos for 15 days. Some embryos had 0.2 ml chicken serum added to the allantoic cavity each day. Although the level of development varied considerably, worms from embryos with added serum developed hind bodies that were substantially larger than those of parasites maintained without added serum. There was no evidence that any worm ingested blood, and only 1 individual, from the serum-augmented group, became ovigerous.     

New synthetic medium for the culture of premature barley embryos

Summary The preparative procedure is presented for a new, synthetic plant tissue culture medium designed specifically for the culture of excised barley embryos. The medium appears capable of supporting development of premature barley embryos with high survivability.     

Aggregation of homozygous Brachyury (T) cells in the culture supernatant of wild-type or mutant embryos

Dissociated cells from wild-type or homozygous mutant (T/T) embryos of mice were cultured in the culture supernatant of +/+ or T/T embryos on a gyratory shaker. The aggregation was promoted by the culture supernatant in the following combinations; +/+ cells in +/+ culture supernatant, +/+ cells in T/T supernatant and T/T cells in +/+ supernatant. When T/T cells were cultured in T/T supernatant, however, only a slight promotion of the aggregation was observed.     

Survival of rabbit embryos after culture or culture/freezing

Five-hundred-and-ninety-five rabbit embryos at the 2- to 4-cell stage were cultured for 48 h to the morula stage. One-hundred-and-sixty-three embryos were transferred directly after culture while the rest (432) were frozen to −196°C. The development of these embryos was tested by transfer into synchronized pseudopregnant recipients or into pseudopregnant recipients 24 h before synchrony. The results were determined at day 17 of pregnancy. The transfer of cultured embryos into synchronized recipients gave a higher survival rate than transfer into asynchronized recipients (51 vs. 15%; P<0.05). The freezing of cultured embryos affected in vitro and in vivo development. Only 56% of the frozen-thawed morulae developed to the blastocyst stage compared with 89% in the control group (P<0.005). The survival rate after synchronous transfer was only 14%. Our results indicate that rabbit embryos need asynchronous conditions when they are frozen and cultured. Embryo survival rate was enhanced by 38% (P<0.07) when these cultured frozen-thawed embryos were transferred into pseudopregnant recipients in an earlier physiological stage (−24 h).     

In vitro development of goat-sheep and goat-goat zona-free cloned embryos in different culture media

The gradual decline in the genetic diversity of farm animals has threatened their survival and risk of their extinction has increased many fold in the recent past. Endangered species could be rescued using interspecies embryo production. The objective of this study was to investigate the effect of three different culture media on the development of Handmade cloned intraspecies (goat-goat) and interspecies (goat-sheep) embryo reconstructs. Research vitro cleave media (RVCL) yielded higher cleavage and morula-blastocyst development in intraspecies and interspecies nuclear transfer groups compared with G1.G2 and modified synthetic oviductal fluid (mSOFaaci). Cleavage frequency of intraspecies cloned embryos in RVCL, mSOFaaci, and G1.G2 did not differ significantly (87.12%, 82.45%, and 92.52%, respectively). However, the morula/blastocyst frequency in RVCL was greater in mSOFaaci and G1.G2 (51.18% vs. 38.28% vs. 36.50%, respectively). Cleavage and morula/blastocyst frequency in interspecies cloned embryos was greater in RVCL than in mSOFaaci and G1.G2 (76.14% and 42.3% vs. 65.9% and 38.3% vs. 58.56% and 33.1%, respectively). Goat oocytes were parthenogenetically activated and cultured in RVCL, mSOFaaci, and G1.G2 and kept as control. Cleavage and morula/blastocyst frequency in this group was greater in RVCL than in mSOFaaci and G1.G2 (89.66% and 65.26% vs. 85.44% and 48.05% vs. 86.58% and 42.06%, respectively). Conclusively, the results suggest that not only can the interspecies embryos of goat be produced using sheep oocytes as donor cytoplast but also the percentages can be improved by using RVCL media for culturing of the embryos.     

Two-phase chemically defined culture system for preimplantation rat embryos

A limiting factor in the development of new technologies and transport of rats worldwide has been the inability to robustly culture preimplantation embryos. Previously, culture in vitro to the blastocyst stage from one-cell embryos was successful only if the one-cell embryos were isolated near the time of the first cleavage and from only a few strains. Here we report the use of commonly available, chemically defined culture media to overcome these limitations. In vitro culture of young one-cell embryos using common embryo media (KSOM, BMOC, or HTF) for 18-22 h followed by culture in mR1ECM medium allows the successful in vitro development of blastocysts from one-cell embryos after 5 days from both outbred (SD) and inbred strains of rat (WF, LEW, F344, and PVG). This system allows the parthenogenetic development of chemically activated, unfertilized oocytes to the blastocyst stage. Embryos cultured in this system develop to term and are live-born following transfer to surrogate mothers.     

A new method for explanting early postimplantation rat embryos for culture

Rat embryos were explanted either on day 8, 3 PM (primitive streak stage) or on day 9, 8 AM (presomite stage) and divided randomly into two groups (plug day = day 0). In one group, Reichert's membrane was removed starting from the side opposite to the ectoplacental cone which was left intact (standard method). In the second group, Reichert's membrane was removed starting from the same side as the ectoplacental cone after the latter structure was excised and the proamniotic cavity (day 8) or the ectoplacental cavity (day 9) destroyed (new method). Embryos were then cultured and examined at 10 AM on day 11. All embryos developed normally in size and general morphology. However, there was a high incidence of inverted heart, allantoic placenta and tail in all groups, except for day 9, new method group; this was the only group in which the ectoplacental cavity did not expand abnormally during culture. The new method, which is quick and easy to perform even when Reichert's membrane does not protrude well, lessens the chances of damaging embryonic ectoderm during the removal of Reichert's membrane. Furthermore, the absence of ectoplacental cone decreases embryo aggregation during culture. We hypothesize that the abnormally expanded ectoplacental cavity generates abnormal physical forces within the egg cylinder that disrupt the control of body asymmetry.     

New serum-free in vitro culture technique for midgestation mouse embryos

Current in vitro culture methods for mouse embryos are critically dependent on specially prepared rodent serum. Rodent serum requires careful preparation and stringent assessment of serum quality, while commercially available whole embryo culture serum is expensive and shows considerable lot variability. Thus, preparation and testing of suitable serum represents a considerable investment of time and resources, particularly for laboratories with only short-term embryo culture requirements. In addition, serum supplementation of culture medium may introduce unknown serum components that could interfere with interpretation of experimental results, especially where the study is geared towards analysis of a specific growth factor. Here we describe the composition of a standardized serum free culture medium comprised of commercially available stem cell media supplements. With this method, we have successfully cultured midgestation stage mouse embryos and demonstrated, using both morphological and gene expression criteria, that these embryos exhibited proper developmental progression. We believe this method to be a significant advance in whole embryo culture technology that will be of particular use to laboratories needing to utilize whole embryo culture to study midgestation organogenesis.     

Use of a uterine-cell monolayer culture system for micromanipulated bovine embryos

The objective of this study was to evaluate the growth of micromanipulated bovine embryos in two in vitro culture systems. Sixty ova (day 7 from estrus) were collected in Dulbecco's phosphate-buffered saline (PBS), with 2% fetal calf serum, and transferred to a PBS holding medium containing 10% fetal calf serum to prepare for micromanipulation. Forty embryos (morula to expanded blastocyst stages) were selected for embryo splitting using a modified microsurgery procedure. Thirty-nine of these embryos were successfully bisected into demi-embryos (DE) and the halves allotted by post-manipulation quality grades into one of two treatment groups (Trt). DE in Trt A were cultured in Ham's F-10 medium with 10% FCS (HF-10) while the remaining DE halves from each embryo were cocultured in HF-10 on a monolayer of endometrial fibroblasts (8 x 10(4) viable fibroblast cells plated three days prior to culture) in Trt B. Embryo development, recorded at 12-hour intervals, was evaluated by a split-plot analysis of variance. Results indicated that embryo viability decreased (P<0.001) over time in culture. Overall viability was greater (P<0.001) for DE in Trt B than in Trt A, with a significant (P<0.05) Trt x Time interaction, indicating that embryo viability decreased more rapidly across time in HF-10 than in the monolayer coculture system. The percentage of DE developing at 12, 24, 36, 48, 60 and 72 hours in culture was: 44%, 41%, 33%, 28%, 21% and 18% for Trt A and 69%, 69%, 69%, 67%, 62% and 62% for Trt B. Fourteen of the DE in Trt B attached to fibroblast monolayer and initiated trophoblastic outgrowth and four additional DE remained viable for up to 17.5 days in vitro as intact blastocysts. These findings are the first reported that demonstrate that the zona-free bovine DE will develop during in vitro culture. Also, the bovine endometrial fibroblast monolayer system proved to be excellent for both short term (相似文献   

New suspension culture system (Mini-AR).

A new suspension culture system (Mini-AR) based on the Stoke's drag law for suspended particles is described. This apparatus can be utilized for the maintenance of mammalian kidney cells in both short- and long-term cultures.     

Metal mesh vitrification (MMV) method for cryopreservation of porcine embryos

The objective was to develop a simpler, more reliable vitrification method for porcine embryos. Prepubertal donor gilts were induced to ovulate with eCG and hCG, and then inseminated artificially. Morulae and expanding blastocysts approximately 200 microm in diameter were collected 6 or 7d after hCG treatment. Embryos collected from donor gilts were maintained, so as to be individually recognizable, and handled in batches of four or five. The embryos together with a minimum volume (<2 microL) of vitrification solution were placed onto stainless steel metal meshes or plastic plates, and then plunged into liquid nitrogen-metal mesh vitrification (MMV) and plastic plate vitrification (PPV), respectively. The meshes or plates were stored in 1.8-mL cryotubes submerged in liquid nitrogen. Stored embryos were subsequently removed, cultured in medium for 24 h, and then assessed for viability. The survival rate (84.4%) of expanding blastocysts cooled by MMV was higher than that (53.1%) of embryos cooled by PPV (P<0.05). There was no significant difference in total cell number between MMV and PPV. The survival rate of morulae cooled by MMV was 55.0%. Transfer of 200 expanding blastocysts cooled by MMV to 10 synchronized recipient gilts resulted in 37 live piglets from 7 recipients. In conclusion, the MMV method was an effective vitrification procedure for cryopreservation of expanding porcine blastocysts. However, there was a batch effect on embryo survival after vitrification.     

Establishment of a reproducible tissue culture system for the induction of Arabidopsis somatic embryos

Somatic embryogenesis is an example of totipotency and is used as a model system for studying embryogenesis. A reproducible tissue culture system was established for the large-scale induction of Arabidopsis somatic embryos. The method allows maintenance of high embryogenic competence over a one-year period. Using this tissue culture system, the expression of embryo-specific genes (ABI3, LEC1, FUS3) was detected in embryogenic cells and somatic embryos. Exogenous application of abscisic acid enhanced the expression of some late-embryogenesis-abundant (LEA) protein genes in somatic embryos. The experiments show that the method can be used to obtain sufficient amounts of embryogenic material for basic molecular analyses.     

Post hatching development: a novel system for extended in vitro culture of bovine embryos

Although acceptable rates of blastocyst formation are achieved with in vitro production of bovine embryos, several problems still compromise the subsequent development of the fetus and newborn, especially in embryos originating from somatic cell nuclear transfer. Routinely, the potential development of a bovine conceptus is predicted either on blastocyst quality or on various parameters related to the embryonic-fetal development in a foster mother. These methods are either imprecise or costly, highlighting the need for more reliable and practical methods to evaluate early embryonic development and differentiation. Thus, our aim was to improve the in vitro culture of embryos post hatching and to define a stable and repeatable system to monitor the development of bovine embryos. For that, in vitro-derived embryos were cultured in agarose gel tunnels in a modified culture medium (SOFaaci within 10% fetal bovine serum and 27.7 mM glucose). Daily monitoring of embryo length revealed that 56%-67% of the embryos in culture showed rapid growth and elongated until Day 13. Electron microscopy of elongated embryos at Day 14 confirmed successful localization of differentiated cells forming the trophoblast and hypoblast, with the definition of the Rauber layer. In conclusion, a stable culture system of post hatching embryos was first defined and can be used as a model for rapid growth, elongation, and initial differentiation of bovine post hatching embryos produced entirely in vitro.     

Improved in vitro development rates of sheep somatic nuclear transfer embryos by using a reverse-order zona-free cloning method

This paper describes a modified zona-free cloning protocol for examining the effects of short-term exposure of donor nuclei to maternal chromosomal components and associated factors. In vitro matured zona-free sheep oocytes were enucleated by micromanipulator-assisted aspiration either before or after fusion with adult fibroblast or granulosa donor cells. Subsequent kinetics of donor nuclei and maternal chromatin as well as in vitro embryo development rates were recorded. The effect of an additional activation stimulus in connection with the reverse-order cloning (fusion before enucleation) and the feasibility of manual enucleation by metal blade were also studied. As a result of the simultaneous fusion and activation, most donor nuclei remained in interphase but swelled in size. Maternal chromosomes reached anaphase II-telophase II stages within 1-2 h of activation, effectively facilitating telophase enucleation with both the micromanipulator-assisted aspiration and manual bisection. A significantly higher development rate to the blastocyst stage was achieved with the reverse-order protocol, suggesting further investigation into the possible role of oocyte nucleus-associated factors in reprogramming is warranted. Overall, the reverse-order zona-free cloning method was efficient in the production of transferable cloned sheep blastocysts and may offer yet another choice of methodology in the practical application of nuclear transfer technology.     

An improved method for isolation of fertile zona-free mouse eggs

Studies of sperm-egg fusion using zona-free mouse eggs are impaired by the procedures used for removal of the zona pellucida. Methods involving proteolytic digestion or mechanical aspiration through micropipettes are limited in that proteases can adversely affect fertility and mechanical removal often results in low egg yields. An efficient procedure for preparation of zona-free mouse eggs was developed using a combined enzymatic (chymotrypsin) mechanical approach (CT-M procedure). Zona-intact eggs, obtained after hyaluronidase treatment, were exposed to 0.001% α-chymotrypsin in medium containing 0.5% bovine serum albumin (BSA). Brief (2 minute) exposure to chymotrypsin under these conditions caused pronounced zona distention in a majority (80-90%) of the eggs, facilitating mechanical removal and resulting in a high yield of zona-free eggs. Eggs prepared by the CT-M method displayed identical penetration levels relative to mechanically denuded eggs. CT-M prepared eggs also showed sperm concentration dependent penetration levels and demonstrated a plasma membrane block to polyspermy, qualities previously observed in mechanically prepared eggs [Wolf DP, 1978, Dev Biol 64:1–10]. Eggs could be exposed to 0.001% CT for zona distention over a 2-10-minute time period with no detrimental effects on fertility. The effect of chymotrypsin was also studied by treating zona-free eggs for 30 minutes over a 1-1,000–μg/ml range of enzyme, and a concentration-dependent reduction in penetration levels was observed. These results indicate that the CT-M method is a useful procedure for the isolation of large numbers of zona-free mouse eggs.     

Primary cell culture from embryos of the Japanese scallop Mizuchopecten yessoensis (Bivalvia)

Primary cell cultures obtained from embryos of Mizuchopecten yessoensis (Bivalvia) survived for four months. Although the number of cells progressively decreased during the cultivation, mitotic cells were observed both at the first stages and at the end. A possibility of growing marine invertebrates cells in long term primary culture is discussed.