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PubMed2003

Effect of activation treatments of recipient oocytes on subsequent development of bovine nuclear transfer embryos.

Effects of recipient oocyte activation methods on the development of nuclear transfer (NT) embryos were investigated. In Exp. 1, cell-cycle phase of serum-starved bovine cumulus cells was examined by flow cytometry. Majority (95.5%) of medium-sized (16-20 microm) cells that made up 56% of total cells was at the G0/G1 phase. NT embryos were constructed by electric fusion with the medium-sized serum-starved cumulus cells and bovine oocytes of 3 different preparations: enucleated oocytes treated with calcium ionophore A 23187 for 5 min and cycloheximide for 5 hr (A 23187/CHX), those treated with ethanol for 7 min and cycloheximide for 2 hr(ethanol/CHX) and those without treatment. In Exp. 2 and 3, developmental competence of NT embryos constructed with A 23187/CHX- and ethanol/CHX-treated oocytes was compared to that of NT embryos constructed with non-treated oocytes, respectively. Further, nuclear behavior in 3 different NT embryos was examined in Exp. 4. Within 1 hr after fusion, majority of the NT embryos constructed with non-treated oocytes showed condensed chromosome. Three hours after fusion, about 50% of NT embryos constructed with non-treated or ethanol/CHX-treated oocytes showed a single pronucleus-like structure. NT embryos constructed with ethanol/CHX-treated oocytes showed similar rates of fusion, cleavage and blastocyst formation to those of the non-treated oocytes. In contrast, NT embryos constructed with A 23187/CHX-treated oocytes did not show any pronucleus-like structure and showed lower cleavage rate and no development to blastocysts. The results indicate that ethanol/CHX-treated oocytes could support development of somatic cell NT embryos to the blastocyst stage at a similar rate to that of non-treated oocytes.

Animals↗
PubMed2003

Morphological assessment of preimplantation embryo quality in cattle.

The extensive use of embryo technologies has emphasized the need for assessing embryo quality by morphological techniques, such as transmission electron microscopy, immunocytochemistry for confocal laser scanning microscopy and fluorescence in situ hybridization. By a combination of these techniques, it has been possible to demonstrate: (i) that rRNA gene activation, as monitored by embryonic nucleolar development, is comparable in bovine embryos developed in vivo and produced in vitro, whereas reconstructed nuclear transfer embryos may be deviant, (ii) that generating embryos by both in vitro production and reconstruction by nuclear transfer is associated with increased occurrence of apoptosis, in particular in the inner cell mass of blastocysts, and (iii) that these two embryo production techniques are associated with increased occurrence of mixoploidy that is, embryos presenting a large population of normal diploid cells and a small population of abnormal haploid or polyploid cells. It is clear that blastocysts that appear healthy at stereomicroscopy may have subcellular defects. Therefore, the possibility of long-term evaluation in vitro of embryos after hatching has been examined. However, whereas embryos developing in vivo after hatching present a number of well defined developmental milestones, such as elongation of the trophoblast, formation of hypoblast and epiblast followed by differentiation of endoderm, mesoderm and ectoderm, in vitro culture systems for development beyond the blastocyst stage currently allow the embryo to complete only a single milestone, namely hypoblast formation.

Animals↗
PubMed2005

Multipotential hematopoietic progenitor cells from embryos developed in vitro engraft unconditioned W41/W41 neonatal miceB.

BACKGROUND AND OBJECTIVES: The first hematopoietic stem cells (HSC) in the mouse able to give rise to the adult hematopoietic system emerge at embryonic day (E) 10.5 in the intraembryonic aorta-gonads-mesonephros (AGM) region, as demonstrated by transplantation into irradiated adult recipients. It has been shown by transplantation into conditioned neonatal or hematopoietic mutant adult recipients that less potent multipotential hematopoietic progenitors exist in the mouse embryo at E9, one day earlier than the appearance of HSC. Studies of the lineage relationships of multipotential hematopoietic progenitors and HSC in the mouse embryo have been complicated by inaccessibility due to in utero development. Attempts are being made to create an in vitro whole mouse embryo culture system to access the developing mouse embryo for such studies of hematopoietic cell emergence during early and mid-gestational stages. The aim of this study was to compare the development of multipotential hematopoietic progenitors in early in utero and in vitro-developed mouse embryos. DESIGN AND METHODS: To test hematopoietic progentior/stem cell activity in the mouse embryonic tissues obtained from genetically marked in utero and in vitro-developed embryos, transplantations were performed using unconditioned neonatal W41/W41 (c-kit hematopoietic mutant) recipients. Long-term donor-cell reconstitution in transplanted mice was measured by (i) semiquantitative polymerase chain reaction and (ii) flow cytometry on peripheral blood and hematopoietic organs. RESULTS: Our experimental data show that multipotent hematopoietic progenitors from in utero-developed embryos engraft unconditioned W41/W41 neonates. Furthermore, in vitro-developed whole embryos also contain early multipotent hematopoietic progenitor cells that are able to yield high-level, long-term engraftment of W41/W41 neonates. INTERPRETATION AND CONCLUSIONS: The in vitro culture of whole mouse embryos during mid-gestational stages allows for the normal growth of multipotential hematopoietic progenitors that can be assayed by transplantation into W41/W41 neonatal recipients. Thus, in vitro-developed whole embryos can be used with confidence in future studies to examine the lineage relationships of multipotential hematopoietic progenitors and HSC.

Animals↗
PubMed2005

Towards an alternative for the acute fish LC(50) test in chemical assessment: the fish embryo toxicity test goes multi-species -- an update.

After its standardisation at the national level in Germany (DIN 38415-6, 2001, 2001), the 48 h sewage testing assay with zebrafish (Danio rerio) embryos has been submitted for standardisation to ISO. As an alternative to the conventional acute (96 h) fish test, a modified fish embryo test will be submitted to the OECD for chemical testing in late 2005. For this, a protocol originally designed for zebrafish was adapted to fit also the requirements of other OECD species, namely medaka (Oryzias latipes) and fathead minnow (Pimephales promelas). Results document that the transfer of the protocol is possible with only minor modifications. Data obtained from embryo tests with the three species are comparable. Statistical analysis of existing zebrafish embryo toxicity data resulted in the conclusions (1) that there is a reliable correlation between the fish embryo test and the acute fish test, (2) that the confidence belt of the regression line was relatively small, but that the prediction range was relatively wide. The regression thus seems appropriate to describe the relationship between acute fish and embryo LC(50) with good confidence, but is less appropriate as a prediction model. Investigations into oxygen requirements of zebrafish embryos reveal that they adapt to a broad range of oxygen levels and survive at concentrations of 2 mg/l without malformations. Zebrafish embryos can thus be exposed in very small toxicant volumes (100 microl), which is of particular interest for the testing of metabolites. Dechorionation studies with 48 h old zebrafish embryos indicate that the barrier function of the chorion increases with the lipophilicity of the test compound. Finally, examples are given as to how additional endpoints can be incorporated into the fish embryo test protocol to extend its scope, e.g. to sediment toxicity assessment or genotoxicity and mutagenicity testing.

Animal Testing Alternatives↗
PubMedDate not supplied

Effects of brief hypoxia and hyperoxia on tissue trace element levels in the developing chick embryo.

Brief hypoxia or hyperoxia has been shown to affect growth and metabolism of chick embryos during the late stages of development. The objective of this study was to alter the availability of oxygen to chick embryos developing in ovo and to determine the effects on tissue zinc, copper, iron and manganese levels. On day 15 of incubation fertile chicken eggs were divided into three groups: 15% O2 (hypoxic), 60% O2 (hyperoxic) and 21% O2 (normoxic) and incubated under these conditions for 72 h to day 18. Hypoxia reduced embryo, heart, brain and liver wet weights, whereas hyperoxia increased embryo, heart, lung and liver wet weights compared to normoxic controls. Chorioallantoic membrane (CAM) wet weight was increased by hypoxia and reduced by hyperoxia. Livers from hyperoxic embryos contained more zinc, iron and manganese and less copper than livers from hypoxic or normoxic embryos. Tissue concentrations of zinc, copper, iron and manganese were reduced in brains from hyperoxic compared to hypoxic or normoxic embryos. Hyperoxia increased the zinc and copper concentrations in CAM, whereas hypoxia reduced zinc and iron levels. The contents of zinc and copper were increased in hyperoxic compared to normoxic or hypoxic lungs. Hearts from hyperoxic embryos had more zinc, copper and manganese than hypoxic or normoxic hearts. Hypoxic yolk sac contained more zinc and manganese than hyperoxic or normoxic yolk sac. Except for yolk sac, the trace element content of tissues from normoxic embryos increased from day 15 to day 18 of incubation in concert with tissue growth. We conclude that the availability of oxygen to the developing chick embryo affects tissue trace element levels through its effects on tissue growth, as a result of adaptation by specific tissues to different oxygen tensions, or via effects on the regulation of trace element uptake and assimilation by the tissues.

Allantois↗
PubMed1990

Comparison of persistence of seven bovine viruses on bovine embryos following in vitro exposure.

The ability of seven cytopathic strains of bovine viruses to adhere to the zona pellucida of six-to-eight day-old bovine embryos were compared. Embryos were exposed to virus by placing them either in virus suspensions or by culturing them on infected bovine turbinate cultures for 18-24 h. After exposure to bovine virus diarrhea virus (BVDV), infectious bovine rhinotracheitis virus (IBV), bluetongue virus (BTV), pseudorabies virus (PRV), vesicular stomatitis virus (VSV), parainfluenza 3 virus (PI3), or bovine enterovirus virus (BEV), the embryos were tested for virus by culture in bovine turbinate cells and by morphological examination using electron microscopy (EM). A special technique to minimize loss of embryos processed for EM was developed. More embryos had viral particles on the surface of the zona pellucida after exposure to 18-24 hour infected cell cultures than did embryos exposed to viral culture suspensions. The most dramatic finding was that BTV adhered in large numbers to the surface of the zona pellucida of exposed embryos. IBRV, PRV, and VSV comprised an intermediate group, with virions occasionally detected on the surface of exposed embryos after 5 washes. Therefore, extensive washing is required. The PI3 and BEV were easily removed from embryo-exposed virus by washing. BVD was difficult to identify morphologically, making assessment by EM unreliable. There was no evidence that any one of the seven viruses penetrated the intact zona pellucida. Using a micromanipulator, 42 embryos were also directly inoculated through the zona pellucida with +/- 50 picoliters of virus inoculum or medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Effect of administration of prostaglandin F2 alpha on embryo recovery from the uterus on day 5 after ovulation in mares.

Ten mares were used to investigate the effect of administration of prostaglandin F2 alpha on uterine tubal motility, as reflected by embryo recovery from the uterus 5 days after ovulation (day 0). Mares were assigned to 3 groups: group A, uterine flush for embryo recovery on day 7; group B, uterine flush for embryo recovery on day 5; and group C, uterine flush for embryo recovery on day 5, after treatment with prostaglandin F2 alpha (10 mg, IM) on day 3. Each mare was assigned to each group once. Embryo recovery rates for the 3 groups were: A, 6 of 10; B, 2 of 8; and C, 0 of 10. The embryo recovery rate for group C was significantly lower (P less than 0.01) than that for group A. Embryo recovery rate for group B was not significantly different from group A or group C. Administration of prostaglandin on day 3 did not increase embryo recovery rate from the uterus on day 5. Additionally, the 25% embryo recovery rate (2 of 8) for group B mares suggested an earlier time for entry of the embryo into the uterus than has previously been reported.

Animals↗
PubMed1986

[Relation between the quality of cattle embryos after thawing and the pregnancy rate after their transfer].

One hundred and forty-two deep-frozen seven days old bovine embryos were, after thawing and stepwise dilution of cryoprotectant, morphologically evaluated under the stereomicroscope. According to the morphology the embryos were divided into four categories as follows: I--fully expanded blastocysts, II--slightly contracted embryos with a minute part of degenerated cells, III--contracted embryos with some degenerated cells, IV--degenerated embryos and those with heavy-damaged zona pellucida, or its complete loss. After surgical transfer of 37 embryos of class I, 16 (43.2%) recipients were pregnant. Transfer of 53 embryos of class II established 20 (37.7%) pregnancies. After transfer of 28 embryos of class III, 10 (35.7%) recipients were pregnant. Twenty four (17%) embryos included in class IV were excluded from transfer. These results indicate that simple morphological evaluation of embryo after freezing and thawing secures relatively objective selection in field conditions. At least this method enables to reveal morphological changes which are not compatible with further development.

Animals↗
PubMed1986

Pattern formation in 8-cell composite embryos of Xenopus laevis.

We have shown in defect experiments that an 8-cell embryo of Xenopus laevis consists of three kinds of cells, that is, animal, vegetal dorsal and vegetal ventral cells, and that cells of different kinds are distinctly different in their developmental capacity. Complete pattern formation occurs in any defect embryo which contains at least two animal, one vegetal dorsal and one vegetal ventral cell. In the present transplantation experiments, we replaced one or two cells of one 8-cell embryo by those of another to obtain 29 series of composite embryos, in which the cell composition of an embryo and/or the dorsoventral orientation of individual cells differed from those of a normal 8-cell embryo. The resulting embryos were examined macroscopically when controls reached stage 26 (tailbud stage) and later. The results showed that both the two animal dorsal cells or one vegetal dorsal cell could be replaced by animal ventral cells or a vegetal ventral cell, respectively, without any detectable effect on pattern formation, irrespective of the ventrodorsal direction of the ventral cells. On the other hand, replacement of an animal ventral or a vegetal ventral cell by an animal dorsal or a vegetal dorsal cell, respectively, made most composite embryos twins. Twins were also formed when a left-handed vegetal dorsal cell was replaced by a right-handed counterpart and vice versa. In these composite embryos, the dorsoventral orientation of the transplanted cell was different from that of a resident dorsal cell or cells of a recipient, and several lines of evidence show that the dorsal cell transplanted in an off-axis orientation is responsible for twin formation. Thus, dorsal cells have the capacity to form dorsal axial structures at later stages and this capacity is localized on the dorsal side, and endows the cells with polarity. On the other hand, ventral cells did not have this capacity or polarity, judging from the fact that their orientation had no effect on pattern formation. One vegetal dorsal or ventral cell could be replaced by an animal dorsal or ventral cell, respectively, without any marked effect. However, replacement of two vegetal cells by animal ones and of one or two animal cells by vegetal ones resulted in deficiency of vegetal cells and oedema and in deficiency of animal cells and incomplete invagination, respectively. Twin formation in composite embryos with animal dorsal cells in place of animal ventral ones is discussed in consideration of findings in recombination experiments by Nieuwkoop.

Animals↗
PubMed1986

The role of ultimobranchial bodies in the modulation of the response of chick embryos to 1,25-dihydroxy-cholecalciferol.

Ultimobranchial bodies (UBBs) were dissected from 17-day-old chick embryos and grafted onto the chorioallantoic membrane of 8-day-old embryos. The embryos with UBB grafts as well as sham-grafted controls were injected on the 10th day of incubation with 100 ng 1,25(OH)2D3 dissolved in ethyl alcohol or with an equal volume of ethyl alcohol alone; embryos were sacrificed on the 13th day. Grafted UBBs showed ultrastructural characteristics typical of actively secreting glands. A histological study of the tibiae from all embryos showed that while the grafted embryos responded to the injection of 1,25(OH)2D3 with a peripheral rim of undermineralized bone trabeculae, sham-grafted embryos never did so. These results confirm the original hypothesis that the presence of differentiated UBBs is a precondition for the production of undermineralized bone (osteoid) by 1,25(OH)2D3. In a second series of experiments, similarly treated embryos were sacrificed on the 10th, 11th, 12th and 13th day; the levels of calcium and inorganic phosphate were determined in their blood. The injection of 1,25(OH)2D3 produced in all embryos hypercalcaemia and hypophosphataemia. However, the hypophosphataemic response was more prolonged in the embryos with UBB grafts than in sham-grafted ones. These results suggest that the grafted UBBs prolonged the hypophosphataemic response, probably by secreting calcitonin and thus reducing the rate of bone resorption. It is also probable that the prolonged hypophosphataemia produced or contributed to the undermineralization of the peripheral (subperiosteal) trabeculae.

Animals↗
PubMed1985

Culture and storage of pig embryos.

Studies have consistently demonstrated that 4-cell pig embryos can be cultured to the blastocyst stage in a simple salt solution containing bovine serum albumin (BSA). Pig embryos appear to be detrimentally affected by lower levels of lactate and pyruvate than are mouse embryos, but in general their in-vitro requirements are similar. Results from embryos cultured between the 4-cell and blastocyst stages are consistent enough to allow the use of culture for the storage and shipment of pig embryos. However, results obtained after culture and transfer indicate a reduction in viability similar to that observed for cultured cattle and mice embryos. Embryos collected earlier than the 4-cell stage have been difficult to support in vitro beyond one or two cleavage divisions. Pig blastocysts may benefit from serum in their in-vitro environment: lamb, fetal calf and human serum supported continued development but pig serum was detrimental. There are conflicting reports on the efficacy of a more complete medium (Minimum Essential Medium) on the growth of pig embryos. Embryo culture will almost certainly contribute to the application of biotechnology to embryos by providing more appropriate environments for in-vitro manipulations and also, possibly, by providing a system for the early selection of desirable embryonic genotypes.

Animals↗
PubMed1979

Latent effects on in vitro development following cytochalasin B treatment of 8-cell mouse embryos.

Eight-cell mouse embryos when treated with 4.0 microgram/ml cytochalasin B (CB) in vitro undergo a reversible developmental arrest. Upon rinsing of embryos and subsequent culture in control medium, normal morphogenetic processes such as compaction of 8-cell embryos, cavitation, and post-blastocyst attachment and outgrowth are restored. However, the effects of CB on mouse embryos are not completely reversible; latent post-blastocyst defects become increasingly more prevalent as CB treatment duration increases. The present study was conducted to quantitatively determine latent effects of CB on post-blastocyst embryos by comparing their ability to attach and to sustain the growth and differentiation of ICM and trophoblast tissues. Groups of 8-cell embryos were cultured in Brinster's BMOC-3 medium containing 4.0 microgram/ml cytochalasin B for 6, 12, 18, and 24 h. Following treatment, embryos were rinsed and cultured until 190 h post coitum (h.p.c.) in Eagle's MEM/10% fetal calf serum modified to contain optimal levels of essential amino acids. Blastocysts generally attached to the surface of the plastic substratum by 120 h.p.c. At selected time periods after attachment (130, 160, and 190 h.p.c.), embryos were scored for outgrowth size, ICM size, extent of peripheral hyaloplasmic fan, and number of trophoblast nuclei per outgrowth. Analyses of variance (ANOVAs) were conducted for each of the four parameters listed above. Rates of attachment were analyzed by chi2 test. Results show that the treatments affect (P less than 0.01) embryo attachment, number of trophoblast nuclei per outgrowth, hyaloplasmic fan production, and ICM growth in a duration-dependent manner. Interestingly, since treatment effects on outgrowth areas are nonsignificant apparently CB does not significantly change total outgrowth area. But CB treatment does cause abnormal fan production and decreased trophoblast nuclei numbers. However, trophoblast cells are apparently more resistant than ICM to CB as is evident by the high incidence of trophoblast outgrowths devoid of ICM. CB (4.0 microgram/ml) treatments at 8-cell stages for relatively short durations (6 and 12 h) induce latent effects on post-blastocyst embryos. Finally, there exists a definite 4.0 microgram/ml CB duration response over the 68-190 h.p.c. observation interval.

Animals↗
PubMed1977

[A contribution to infection with yellow fever virus 17D in chick embryos (author's transl)].

During the production of yellow fever virus 17 D vaccine from chick embryos, few embryos die in the time between injection of the eggs and harvest of the embryos. In order to answer the question whether the death of the embryos is due to the infection or to the injury caused by the injection, it is necessary to examine the embryos macro- and microscopically for pathological changes. 8 and 9 days old chick embryos were inoculated into the amniotic cavity with different concentrations of yellow fever virus 17 D (table 1). The embryos were removed from the eggs between the 2nd and 7th day after inoculation. Heart, lung, kidney, brain, liver, and spleen were removed. Macroscopically observable pathological symptoms of the embryos and their organs were recorded. Sections of the organs were histologically investigated. Some embryos died soon after the infection, others on the 5th and 6th day of incubation (table 2). Their death was dependent on the dosage of the inoculum. Macroscopically, some embryos showed oedemas, petechiae on head and trunk, haemorrhages of liver and kidney, enlargement of liver and spleen, and a yellow discolouration of the liver (table 3). Microscopically, liver and brain showed the greatest pathological changes; heart and kidney were also affected, whereas lungs and spleen seemed to be unaffected. The following pathological changes were observed: slight fatty degeneration, oedemas, vascular inflammation, perivascular infiltrates, diffuse infiltrations, infiltrations in form of small nodules and necroses (table 3 and figures 1-12). the severity of the symptoms was evaluated using arbitrary units. These units are summarized on table 3 according to time of occurrence p.i., to virus dilution, to organ, and to type of symptom. An index of pathological changes was allived by dividing the sum of units by the number of organs. The maximum value of the index was demonstrable in liver and brain (table 4). Figure 13 adn table 5 show the development of the lesions during the infection. The maximum value of the index was reached on the 5th day p.i. The most significant pathological changes, as necrosis and perivascular infiltrate, were mainly observed from the 5th day p.i. on (table 6). The perivascular infiltrates were found in heart and brain, the necroses in brain and liver (table 7).

Animals↗
PubMed1994

In vitro viability of cryopreserved equine embryos following different freezing protocols.

The main objective of this study was to evaluate two freezing protocols and the effect of agar embedding on survival of day 6.5 equine embryos. A total of 133 embryos were used, in one group (n = 51), embryos were first embedded in agar before the freezing protocol was started. A freezing protocol to -30 degrees C or -33 degrees C was used before plunging embryos into liquid nitrogen (LN2). The embryos were thawed in water at 37 degrees C, evaluated and placed in culture. After 24 h culture, the embryos were evaluated for their morphology and development. No differences were observed between embryos plunged at -30 degrees or at -33 degrees C in LN2. The analysis of the morphology and development after thawing showed that the diameter and developmental stage at freezing correlated with embryo survival. Morula and early blastocyst stages of development were associated with better quality after freezing and thawing and had a better potential to survive after in vitro culture (p < 0.05) compared to more advanced stages. The agar failed to protect embryos from zona pellucida damage, but a tendency to prevent rupture was observed in larger embedded embryos.

Animals↗
PubMed1993

A multifactorial analysis of the pregnancy outcome in a successful embryo cryopreservation program.

OBJECTIVE: To determine which factor, or factors, may influence the success of an embryo-freezing program. DESIGN: Prospective. SETTING: An established, private IVF clinic. PATIENTS: This study comprises data from 185 consecutive thaw cycles (161 patients) that resulted in embryo replacement. The influence of the following factors, with regard to pregnancy outcome, was specifically addressed: embryo quality, day of the endogenous LH surge, number of thawed embryos replaced, length of time frozen, E2 level at the time of the hCG trigger in the stimulated cycle, and patient age at the time of freezing. RESULTS: An overall pregnancy rate (PR) of 27.6% per ET was achieved in this study. Regardless of the day of the LH surge, patients who had at least one good quality embryo (the "sponsoring embryo") conceived 35% of the time. Patients who lacked a sponsoring embryo frozen conceived 15% of the time. Patients who were 39 years of age or younger had a PR of 31% per transfer. None of the 22 patients who were 40 or older conceived. No other factor examined had any effect on pregnancy outcome. CONCLUSIONS: Good quality frozen/thawed embryos are a major factor in achieving a consistently high PR. Poorer quality embryos are capable of implantation but at a significantly reduced rate. Patient age was also found to be significant in predicting pregnancy. Neither the day of the LH surge, number of embryos transferred, length of time frozen, nor the E2 level appears to have any effect on the establishment of pregnancy.

Adult↗
PubMed1996

Aneuploidy 16 in human embryos increases significantly with maternal age.

OBJECTIVE: To determine aneuploidy for chromosome 16 by recycling nuclei of cells already analyzed for chromosomes X, Y, 18, 13, and 21 using multiple fluorescence in situ hybridization in preimplantation human embryos in a time frame compatible with clinical IVF and to asssess the incidence of chromosome 16 aneuploidy in embryos related to maternal age. DESIGN: Prospective experimental study. SETTING: In vitro fertilization program in a tertiary center. PATIENTS: One hundred four consenting patients undergoing IVF. MAIN OUTCOME MEASURES: Chromosome 16 ploidy was analyzed in a total of 195 embryos. In 89 embryos, a standard multiple-probe fluorescence in situ hybridization was used for chromosomes X, Y, 18 and 16 (series 1). The remaining 106 embryos (series 2) were reanalyzed with a new procedure for chromosome 16, which involves rehybridization with a digoxigenin-labeled alpha satellite probe after the standard analysis for chromosomes X, Y, 18, 13, and 21 was completed. The embryos were assigned to one of three groups according to the women's age; group 1: </= 34 years (n = 34), group 2: 35 to 39 years (n = 47); group 3: >/= 40 years (n = 23). RESULTS: Successful analysis, including biopsy, fixation, and fluorescence in situ hybridization was achieved in 86% of the blastomeres within approximately 10 hours. A significant relationship was found between the rate of aneuploidy for chromosome 16 and increasing maternal age: group 1: 0%, group 2: 6.3%, and group 3: 11.7%. Monosomy for chromosome 16 was found in 72.7% of the 11 embryos carrying chromosome 16 anomalies, with the remaining three embryos having two trisomies and one tetrasomy. This new protocol was applied clinically to five patients undergoing preimplantation aneuploidy assessment. Aneuploidy for chromosome 16 was found in five embryos from three of those patients. CONCLUSIONS: This study demonstrates that preimplantation genetic diagnosis of the major human aneuploidies is achievable within a time frame compatible with IVF. In addition, this study confirms, for embryos, the existing data from spontaneous abortions suggesting that chromosome 16 aneuploidy increases with maternal age. The high prevalence of embryonic monosomy, which is rarely found in spontaneous abortions, suggests that monosomy 16 could be a factor associated with failure of implantation, as well as pointing to a different mechanism involved in the generation of chromosome 16 aneuploidy.

Adult↗
PubMed1996

Recovery rate and quality of embryos from mares inseminated at the first post-partum oestrus.

The pregnancy rate is lower in mares inseminated at the first post-partum (p.p.) oestrus (40-50%) compared with pregnancy rates in subsequent oestrous cycles (55-65%). The causes of the lowered pregnancy rate are not fully understood. The aim of the present study was to examine if embryonic defects could be one of the reasons for lowered pregnancy rate. A total of 23 p.p. and 14 non-lactating control mares were flushed 7 days after detection of ovulation. Embryo recovery rate was 48% and 71% in p.p. and control mares, respectively (p = 0.16). Embryos were photographed, measured, graded and stained with fluorescein diacetate to assess their viability. Thereafter embryos were bisected and stained with Hoechst 33342 to count the cell nuclei. Embryos in both groups were equally viable and the cell numbers were not significantly different. According to morphological evaluation all embryos were classified as excellent or good. Embryos aged 7.3 to 7.6 days (+/-0.25 days) were smaller in the p.p. group than in the control group (p < 0.05). Forty-seven (9/19) and 8% (1/13) of the uterine swabs, taken before the first insemination, yielded bacteria and neutrophils in p.p. and control mares, respectively. The amount of neutrophils and/or bacteria had no statistically significant effect on embryo recovery rate (p > 0.10). Recovery of embryos was not related to histological findings in uterine biopsies taken after embryo recovery. Embryo recovery rate in p.p. mares (48%) was similar to previously reported foal heat pregnancy rates (40-50%). Hence, early embryonic death in utero would not be the most likely reason for lowered pregnancy rate in mares inseminated at the first p.p. oestrus. Sperm transport and oviductal conditions by the time of the first p.p. oestrus would need to be studied to clarify the role of fertilisation failure as the cause of lower pregnancy rate in mares inseminated at foal heat.

Animals↗
PubMed1997

Surgical manipulation of mammalian embryos in vitro.

Whole-embryo culture systems are useful in the fields of not only embryology but also teratology, toxicology, pharmacology, and physiology. Of the many advantages of whole-embryo culture, we focus here on the surgical manipulation of mammalian embryos. Whole-embryo culture allows us to manipulate mammalian embryos, similarly to fish, amphibian and avian embryos. Many surgical experiments have been performed in mammalian embryos in vitro. Such surgical manipulation alters the destiny of morphogenesis of the embryos and can answer many questions concerning developmental issues. As an example of surgical manipulation using whole-embryo culture systems, one of our experiments is described. Microsurgical electrocauterization of the deep preaxial mesodermal programmed cell death zone (fpp) in the footplate prevented the manifestation of polydactyly in genetic polydactyly mouse embryos (Pdn/Pdn), in which fpp was abolished.

Animals↗