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PubMed2003

Successful production of piglets derived from vitrified morulae and early blastocysts using a microdroplet method.

This study was conducted to determine the efficiency of vitrification using the microdroplet (MD) method for early stage porcine embryos. Embryos at compacted morulae to early blastocyst stage were vitrified in a vitrification solution containing 40% (v/v) ethylene glycol, 0.6M sucrose and 2% (w/v) polyethylene glycol in M2 (ESP) without any pretreatment. The equilibration and dilution were carried out in third and fourth steps, respectively, at 38 degrees C. The survivability of the cryopreserved embryos was assessed for both in vitro culture (Experiment 1) and by embryo transfer (Experiment 2). In Experiment 1, the embryos were vitrified within a microdroplet or 0.25 ml straw (ST) and fresh embryos were used as a control group. The survival rates after 24h culture in the MD, ST and control groups were 21/23, 14/20 and 20/20, respectively. The hatching rates of the embryos after 48 h incubation were 14/23, 4/20 and 16/20, respectively. In Experiment 2, 171 vitrified embryos were transferred to 5 recipient gilts, and 17 healthy piglets were produced from 2 recipients (3 recipients aborted) in Group 1. In Group 2, 81 vitrified embryos and 16 fresh embryos in total were transferred to 4 recipient gilts, and 10 healthy piglets from the vitrified embryos were produced from 3 recipients. These results indicated that porcine embryos of compacted morulae to early blastocyst stage can survive cryopreservation using the microdroplet method without any special intracellular manipulation or treatment.

Animals↗
PubMed1990

Developmental capacity of mechanically bisected mouse morulae and blastocysts.

Mouse embryos were mechanically bisected at the morula, early blastocyst or expanded blastocyst stages of development and cultured in vitro to the expanded blastocyst stage. Their capacity for postimplantation development was assessed after transfer to pseudopregnant foster mice. Embryos bisected at blastocyst stages had a higher survival rate in vitro than those bisected at the morula stage. Half-embryos had approximately half the number of cells at the blastocyst stage as control embryos, but the proportion of cells in the inner cell mass (ICM) was unaltered. The implantation rate of blastocysts derived from bisected embryos was only slightly lower than that of control embryos, but bisected embryos had a significantly reduced capacity to form fetuses. Histological analyses showed that failure to form a fetus is due to the absence of egg cylinder development, which correlates with the reduced number of cells in the ICM of bisected embryos. Postimplantation viability of half-embryos was significantly higher when blastocysts were transferred to Day-3 rather than Day-4 pseudopregnant recipients, presumably because of an increase in cell number in vivo prior to implantation.

Animals↗
PubMed2002

Births after vitrification at morula and blastocyst stages: effect of artificial reduction of the blastocoelic cavity before vitrification.

BACKGROUND: In 1996, with the introduction of sequential media, we set up a programme of cryopreservation of supernumerary morulae (day 4) and blastocysts (day 5) using a vitrification procedure. Our results showed that the efficiency of the vitrification method was dependent on the stage of embryo development and was negatively correlated with the expansion of the blastocoele. We postulated that a large blastocoele might disturb cryopreservative potential due to ice crystal formation during the cooling step. We analysed therefore the effectiveness of reducing before vitrification the volume of the blastocoelic cavity. METHOD: Day 4 and day 5 embryos were vitrified in 40% ethylene glycol-18% Ficoll and 0.3 mol/l sucrose before plunging the straws directly into liquid nitrogen. Artificial shrinkage of the blastocyst was achieved after pushing a needle into the blastocoele cavity until it contracted. RESULTS: The survival rate post-thawing of day 4 and intact day 5 embryos was correlated with the volume of the blastocoele. In the control group only 20.3% blastocysts or expanded blastocysts survived as compared with 54.5 and 58.5% with morulae and early blastocyst respectively. After puncturing the blastocoelic cavity, an increase in the survival rate of up to 70.6% was noted. The pregnancy rates were improved after the artificial shrinkage procedure (20.5%) compared with the control intact blastocyst group (4.5%) (not significant). After reduction of the blastocoelic cavity, a significant increase in the implantation rate per vitrified blastocyst was observed (12.0 versus 1.4% P < 0.01). CONCLUSIONS: Our results showed that survival rates in cryopreserved expanded blastocysts could be improved by reducing the fluid content. This was presumably because mechanical damage caused by ice crystal formation was avoided. These observations should be considered when establishing a strategy and a protocol for cryopreservation of day 5 embryos.

Adult↗
PubMed1993

Assessment of polyploidy in human morulae and blastocysts using co-culture and fluorescent in-situ hybridization.

Fluorescence in-situ hybridization with DNA probes for X, Y and no. 18 chromosomes was used to analyse human morulae (n = 13) and blastocysts (n = 41), obtained after co-culture on Vero cells. On the basis of the number of hybridization signals, the proportion of embryos with more than five polyploid cells was 30.8% for morulae and 29.3% for blastocysts. These values are similar to those for mixoploidy (mosaicism of diploid and polyploid cells) observed in blastocysts of animal species. The results were confirmed by scanning electron microscopy, which showed a wide variation in the size of blastocyst nuclei, and by classical cytogenetic analysis. Mixoploidy seems to be a normal feature in preimplantation embryos and to occur very early in human embryo development. This lays open to doubt the preimplantation diagnosis of genetic errors at these stages, since results obtained from single cell analysis may not be representative of the whole embryo.

Animals↗
PubMed1999

Developmentally regulated loss and reappearance of immunoreactive somatic histone H1 on chromatin of bovine morula-stage nuclei following transplantation into oocytes.

One difference between chromatin of bovine oocytes and blastomeres is that somatic subtypes of histone H1 are undetectable in oocytes and are assembled onto embryonic chromatin during the fourth cell cycle. We investigated whether this chromatin modification is reversed when nuclei containing somatic H1 are transplanted into ooplasts. Donor nuclei obtained from morula-stage bovine embryos were fused to ooplasts at different times before and after parthenogenetic activation of the ooplasts. After fusion, immunoreactive H1 became undetectable, and the loss occurred more rapidly when fusion was performed near the time of ooplast activation compared with several hours after activation, when the host oocytes were at a stage corresponding to interphase. Although the loss of immunoreactive H1 occurred independently of DNA replication and transcription, exposure of reconstructed oocytes to cycloheximide or 6-dymethylaminopurine (6-DMAP) delayed the loss of immunoreactive H1 from transplanted nuclei. During further development of nuclear-transplant embryos, somatic H1 remained undetectable at the 2- and 4-cell stages, and it reappeared on the chromatin at the 8- to 16-cell stage, as previously observed in unmanipulated embryos. We conclude that factors in oocyte cytoplasm are able to modify morula chromatin so that somatic H1 becomes undetectable, and that the amount or activity of these factors declines over time in activated ooplasts.

Adenine↗
PubMed2007

Vitrification of boer goat morulae and early blastocysts by straw and open-pulled straw method.

The aim of this study was to investigate the effects of different vitrification solutions [EFS30 or EFS40 contains 30% (v/v) ethylene glycol (EG), 40% (v/v) EG; EDFS30 or EDFS40 contains 15% (v/v) EG and 15% (v/v) dimethyl sulfoxide (DMSO), 20% (v/v) EG and 20% (v/v) DMSO], equilibrium time during vitrification (0.5-2.5 min) and vitrification protocols [one-step straw, two-step straw and open-pulled straw (OPS)] on in vivo development of vitrified Boer goat morulae and blastocysts after embryo transfer. In the one-step straw method, the lambing rates of vitrified embryos in EFS30 (37.5%), EFS40 (40.5%) or EDFS30 (38.2%) group were similar to that of fresh embryos (57.5%) and conventional freezing method (46.7%) when the equilibrium time was 2 min. In the two-step straw method, the highest lambing rate was obtained when embryos were pretreated with 10% EG for 5 min and then exposed to EFS40 for 2 min (51.4%), showing similar lambing rates compared with fresh embryos (56.1%) or the embryos cryopreserved by conventional freezing method (45.2%). In the OPS method, the lambing rate in EFS40, EDFS30 or EDFS40 groups were similar to that (57.1%) of fresh embryos, or to that (46.0%) of embryos cryopreserved by conventional freezing method. The highest lambing rate (51.4%) of the group of OPS was obtained when the embryos were vitrified with EDFS30. In conclusion, either the two-step straw method in which embryos were pretreated in 10% EG for 5 min and then exposed to EFS40 for 2 min, or the OPS method in which embryos were pretreated in 10% EG + 10% DMSO for 30 s and then exposed to EDFS30 for 25 s was a simple and efficient method for the vitrification of Boer goat morulae and blastocysts.

Animals↗
PubMed1985

Multiple embryo-transplant offspring produced from quartering a bovine embryo at the morula stage.

A non-surgical embryo collection was completed on a day 7 superovulated Chianina donor cow. Because all but two of the ova from the collection were unfertilised and a surplus of potential recipients was available, one embryo (an excellent quality late morula) was dissected into four equal portioned 'quarter' embryos using a simplified micromanipulation procedure. Each quarter embryo was then placed in a 0.25 ml French straw and non-surgically transplanted to four different crossbred beef recipient females. The remaining embryo was similarly transplanted to a herd mate recipient as an intact embryo. One recipient returned to oestrus, one recipient had an extended post transfer cycle and the two remaining recipients produced a live quarter embryo transplant calf each within 24 hours of the other. The intact embryo placed in a herd mate recipient did not produce a transplant calf. To the authors' knowledge, these transplant offspring are the first live births reported from a non-surgically collected later-stage bovine morula (day 7), which had been dissected into quarters and then individually transplanted non-surgically to recipient females. The procedure was relatively simple to perform and was completed in less than one hour.

Animals↗
PubMed1982

Effect of post-ovulatory age of recipient on survival of frozen-thawed rabbit morulae.

A significantly higher survival rate was obtained when frozen-thawed morulae were transferred to oviducts of recipients ovulating after donors rather than to those of synchronized or earlier ovulating recipients. The highest proportion of live young, 23%, was obtained after transfer of eggs to recipients ovulating 18 h after donors. The proportion of live young (48%) was significantly increased when frozen-thawed rabbit morulae were transferred into both oviducts of recipients at this stage.

Animals↗
PubMed1984

Degradation of biochemical pools labelled with [14C]glucose during culture of 8-cell and morula--early blastocyst-stage mouse embryos in vitro and in vivo.

When 8-cell mouse embryos were chase cultured for 24 h in vitro or in vivo (in uteri of pseudopregnant mice) there was no indication of utilization of the small amount of acid-soluble glycogen synthesized during the pulse. At the morula-early blastocyst stage of development almost 50% of the label incorporated during the pulse was found in the acid-soluble glycogen fraction. The biochemical pools at this stage were relatively stable in vitro and in vivo during a short (5 h) chase period. However, marked degradation of the acid-soluble glycogen pool occurred during long periods of exposure to the uterine environment and, over 48 h in utero, almost all of the label was lost from this pool. By contrast, embryos cultured in vitro for the same period retained greater than 60% of their acid-soluble glycogen. Utilization of glucose carbon in the acid-insoluble glycogen fraction occurred during in-vitro and in-vivo chase but there was a suggestion that the change in vivo was less than that in vitro. The non-glycogen macromolecular pool was relatively stable except during extended chase culture of morulae-early blastocysts when some utilization occurred. Under these conditions utilization was less in utero than in vitro. The experiments show that the uterine environment has a marked influence on the metabolism, particularly of glycogen, of the embryo and indicate that some factor in the uterus causes net degradation of acid-soluble glycogen by the embryo at the late preimplantation stage of development.

Animals↗
PubMed1990

Development and survival after transfer of cow embryos cultured from 1-2-cells to morulae or blastocysts in rabbit oviducts or in a simple medium with bovine oviduct epithelial cells.

This study compares development of bovine 1-2-cell embryos in bovine oviduct epithelial cell co-culture (Group EC) with a glucose- and serum-free simple medium (CZB), or after surgical transfer to ligated oviducts of rabbits (Group RO). Embryos were surgically collected from superovulated donor cows 40-48 h after the beginning of oestrus and randomly distributed between the two groups. Embryos were cultured or incubated for 5 days. In Exp. 1, embryo quality scores and total numbers of cells in the two groups were compared. In Exp. 2, pairs of similarly treated morulae were transferred to each of 10 or 12 recipients in the Groups RO and EC, respectively. Total cell counts per embryo in both groups averaged 52 (P greater than 0.05), and the in-vitro culture system was equivalent to the rabbit oviducts in promoting embryo development for all characteristics measured. Embryo survival, as determined by ultrasound between Days 39 and 43 after oestrus, in 13 ideal recipients was 57% for embryos in Group EC and 58% for embryos Group RO. None of the 9 less desirable recipients was pregnant for either group. These results establish that cattle zygotes can develop to morulae in culture with bovine oviduct epithelial cells in a simple medium and can produce normal pregnancy rates.

Animals↗
PubMed1982

Observations on the development of bovine morulae on various cellular and noncellular substrata.

The objective was to determine the effect of various cell types on the development of bovine embryos in vitro. Morulae were collected from beef and dairy cows 4 to 5 d after the onset of estrus (d 0). Embryos were randomly allocated to nine treatments: (1) microdrops of Ham's F-10 medium (HF-10) under paraffin oil (HF-10); (2) HF-10 over a substratum of collagen without paraffin oil (HF-10/clg); (3) HF-10 with bovine uterine fibroblasts grown on a collagen substratum (HF-10/clg-fib); (4) Minimal Essential Medium (MEM) without paraffin oil (MEM); (5) microdrops of MEM under paraffin oil (MEM/oil); (6) MEM with bovine uterine fibroblasts (MEM/Buf; (7) MEM with bovine testicular fibroblasts (MEM/Btes); (8) MEM exposed to uterine fibroblasts for 24 h to condition the medium (MEM/Con), and (9) MEM with 1 mm cubes of bovine endometrial tissue (MEM/End). In all cases, media were supplemented with 10% (v/v) heat-treated fetal calf serum. Observations were made at 24 h intervals, with the stage of embryo development recorded. Morulae developed into expanded blastocysts more frequently (P less than .05) in HF-10, MEM, MEM/oil, MEM/Buf, MEM/Btes and MEM/Con than in HF-10/clg, HF-10/clg-fib and MEM/End. More (P less than .01) blastocysts hatched in MEM/Buf and MEM/Btes than in the other media. In addition, nine of 12 (MEM/Buf), eight of 11 (MEM/Btes) and two of two (HF-10/clg-fib) hatched blastocysts attached to the fibroblast monolayers, an observation that was uncommon in treatments without cells. These data suggest that the co-culture of bovine embryos with bovine fibroblasts in a superior system for promoting embryo hatching and attachment in vitro.

Animals↗
PubMed1988

Culture of bovine morulae in media supplemented with immunoglobulins.

Bovine morulae (d 6; n = 257) were obtained to evaluate the effect of Ig on embryo development in vitro to the hatched blastocyst stage or until degeneration. Embryos were cultured in Ham's F-10 containing either 6.4 mg/ml steer serum, .64 mg/ml bovine gamma globulins, bovine IgG, bovine IgM alone, and steer serum added to gamma globulins, IgG, or IgM. In steer serum alone, 69.6% of the embryos developed to the expanded blastocyst stage, whereas 73.9% of embryos in gamma globulin and steer serum and 25% of embryos in gamma globulin alone reached the same stage of development. None of the morulae cultured in IgG and IgM alone or IgG and IgM with steer serum reached the expanded blastocyst stage. Additionally, the times to degeneration were shorter when embryos were cultured in IgG (54.9 h) and IgM (59.4 h) plus steer serum than for embryos placed in gamma globulin plus steer serum (77.6 h) or steer serum (81.5 h) alone. These results suggest that .64 mg/ml bovine gamma globulin did not affect embryo growth, whereas .64 mg/ml IgG and IgM were toxic to early development of bovine embryos in vitro.

Animals↗
PubMedDate not supplied

In vitro viability of vitrified mouse morulae thawed at different temperatures.

The effect of thawing temperature on in vitro development of vitrified mouse morulae was investigated. The embryos were vitrified in a solution based on ethylene glycol as cryoprotectant, and Ficoll as macromolecule to assist vitrification. They were then thawed at 20 degrees, 37 degrees and 48 degrees C for 6 sec and at 48 degrees C for 2 sec. Among groups, there was no significant difference on the development at 72 h of culture when embryos were thawed at 20 degrees, 37 degrees C for 6 sec or 48 degrees C for 2 sec. At 48 h of culture the embryos thawed at lower temperature had a reduced resumption (69.5%) while the embryos thawed at 37 degrees and 48 degrees C for 2 sec had a higher resumption rate (80.0% and 82.5%). It was concluded that a high development in vitro of vitrified mouse morulae can be obtained at three different temperatures of thawing, although at higher temperatures there seems to be a tendency of an earlier resumption development.

Animals↗
PubMed1997

Full-term development after transfer of nuclei from 4-cell and compacted morula stage embryos to enucleated oocytes in the mouse.

The developmental ability of enucleated mouse oocytes reconstituted between the nucleus from 4-cell mouse embryos at different cell stages and recipient cytoplasms at different conditions, and the developmental ability of oocytes receiving nuclei from compacted morulae were examined. The highest development was observed with the nucleus at the G1 stage fused with cytoplasm at the M stage. Although the enucleated oocytes receiving a nucleus from 4-cell embryos did not develop after transfer to a recipient female, young were obtained after renuclear transfer to enucleated fertilized eggs. Live mice were also obtained from the nucleus of compacted morula.

Animals↗
PubMed2000

Origin of the Henze solution/precipitate from morula cells of the blood of the ascidian Phallusia mammillata.

The "Henze solution", derived originally from the aqueous extraction of pelleted whole blood from the ascidian Phallusia mammillata, was examined using spectral studies. The aqueous extraction of fractionated blood cells including compartment cells, signet ring cells, and morula cells obtained using cell separation techniques were also examined. It was found that this Henze solution, and the Henze precipitate itself derived from this solution, emanated solely from the morula cells. Furthermore, it was found that this solution is formed independently of the vanadium metal ions otherwise associated with the vanadocytes. Observation of the Henze precipitate by light microscopy shows that this material partially forms crystallites or microglasses.

Animals↗
PubMed1996

Sucrose-induced shrinkage of in vitro produced bovine morulae: effect on viability, morphology and ease of evaluation.

Sucrose (0.3 M) was used to cause artificial compaction of the embryonic cell mass of in vitro produced bovine embryos to facilitate morphological evaluation. Embryos were produced using routine in vitro maturation (IVM) and fertilization (IVF) techniques. The time necessary to induce shrinkage in 0.3 M sucrose to 75% of the original volume of Day 5 morulae was found to be less than l min, and 95% of the volume was regained in PBS after 2.5 min. No detrimental effect was observed after a 5- to 10-min sucrose treatment on subsequent blastocyst formation at Days 6 and 7 (P > 0.05). Furthermore, no significant differences were observed in the total number of cells, or in the mitotic and pycnotic cell index of blastocysts in different treatment groups. Agreement among 7 evaluators grading 40 Day 6 embryos was examined using the kappa coefficient of agreement (kappa). Overall agreement among evaluators for classification of quality grade was poor (48.2 %, kappa = 0.31) for embryos evaluated in PBS, but the rate improved when the same embryos were scored in sucrose (62.5 %, kappa = 0.49). Evaluating less compact in vitro produced bovine morulae in sucrose increases agreement among evaluators, since embryos in sucrose mimick the appearance of in vivo produced embryos. Thus, we conclude that scoring in vitro produced embryos in sucrose improves agreement among evaluators.

Journal Article↗
PubMed1976

Morula forms of E rosettes: distribution, specific inhibition, and enhancement.

Rosetts-forming cells (RFC) with more than 10 sheep red blood cells (SRBC's) per cell defined as morula-forming cells (MFC) were studied in human lymphoid organs and in the peripheral blood (PBL). MFC are most common in the thymus (more than 50%) and the frequency is much less in peripheral lymphoid tissues and in the blood (29.8% in those less than 5 years old; 14.9% in adults). There is very slight decrease in total RFC in PBL over age 5, but the changes in MFC are quite striking. Treatment with antihuman thymus antiserum (ATS) and with anti-human lymphoblastic antiserum (ATL) resulted in significant inhibition of total rosette and morula rosette formation. No inhibition was seen using normal brain antiserum, anti-IgM antiserum, or anti-L chain antiserum. In two patients with chronic lymphocytic leukemia and in some cases of thymic deficiency the ratio of MFC to total RFC was unusually high. After culture with phytohemagglutinin (PHA) and conversion to blast forms or after surface peturbation by papain or freeze-thaw treatment, MFC numbers were increased.

Humans↗
PubMed1976

Investigation of inner cell mass determination by aggregation of isolated rat inner cell masses with mouse morulae.

Inner cell masses (ICMs) dissected from 4 1/2-day rat blastocysts were aggregated with 2 1/2-day mouse morulae. Successful aggregates formed blastocysts in vitro and morphologically normal 5 1/2-day conceptuses in the mouse uterus. Immunofluorescent analysis of these conceptuses revealed that rat cells were only present in the embryonic ectoderm and endoderm and never in the trophectoderm derivatives, although rat trophoblast did develop in the mouse uterus in various control experiments. The single-cell resolution of this technique extends the results obtained from aggregating mouse ICMs with mouse morulae and provides strong evidence that ICM cells, although not overtly differentiated, are determined by the blastocyst stage.

Animals↗