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PubMed1999

Development to live young from bovine small oocytes after growth, maturation and fertilization in vitro.

Bovine oocytes (90 to 99 microns in diameter) were isolated from early antral follicles (0.5 to 0.7 mm in diameter). Cumulus-oocyte complexes (COC) with pieces of parietal granulosa were embedded in collagen gels and cultured for 14 d. After in vitro growth culture, oocytes recovered from the collagen gels were further matured, fertilized and cultured in vitro, and then were transferred to recipient cows. After 14 d of growth culture, 37% of the oocytes (203/556) showed normal morphology in the collagen gels. The mean diameter of the oocytes was 110.1 +/- 6.0 microns, significantly larger (P < 0.01) than before growth culture (94.8 +/- 2.7 microns), and 77% were at the germinal vesicle stage while 23% had undergone germinal vesicle breakdown. After 24 h of maturation culture followed by insemination, 27% of in vitro-grown oocytes reached the second metaphase, and 42% of the oocytes were normally fertilized. After insemination, 18.2% of in vitro-grown oocytes cleaved and 3.7% developed to the blastocyst stage. Three blastocysts obtained from in vitro-produced 90- to 99-micron oocytes were transferred to 3 recipients. One recipient subsequently became pregnant and delivered a live calf on Day 277. These results demonstrated for the first time that 90 to 99-micron oocytes from early antral follicles can complete growth and acquire full developmental competence in vitro so that live young can be produced after maturation, fertilization, subsequent culture in vitro, and transfer to recipient cows.

Animals↗
PubMed1999

Improved in vitro embryo development using in vivo matured oocytes from heifers superovulated with a controlled preovulatory LH surge.

In bovine in vitro embryo production, the IVM step is rather successful with 80% of the oocytes reaching the MII stage. However, the extent to which the process limits the yield of viable embryos is still largely unknown. Therefore, we compared embryonic developmental capacity during IVC of IVF oocytes which had been matured in vitro with those matured in vivo. In vitro maturation was carried out for 22 h using oocytes (n = 417) obtained from 2- to 8-mm follicles of ovaries collected from a slaughterhouse in M199 with 10% fetal calf serum (FCS), 0.01 IU/mL LH, and 0.01 IU/mL FSH. In vivo matured oocytes (n = 219) were aspirated from preovulatory follicles in eCG/PG/anti-eCG-superovulated heifers 22 h after a fixed time GnRH-induced LH surge; endogenous release of the LH surge was suppressed by a Norgestomet ear implant. This system allowed for the synchronization of the in vitro and in vivo maturation processes and thus for simultaneous IVF of both groups of oocytes. The in vitro developmental potential of in vivo matured oocytes was twice as high (P < 0.01) as that of in vitro matured oocytes, with blastocyst formation and hatching rates 11 d after IVC of 49.3 +/- 6.1 (SEM; n = 10 heifers) vs 26.4 +/- 1.0% (n = 2 replicates), and 39.1 +/- 5.1% vs 20.6 +/- 1.4%, respectively. It is concluded that IVM is a major factor limiting in the in vitro production of viable embryos, although factors such as the lack of normal preovulatory development of IVM oocytes contributed to the observed differences.

Abattoirs↗
PubMed2003

Macrophages in chronic type 2 inflammation have a novel phenotype characterized by the abundant expression of Ym1 and Fizz1 that can be partly replicated in vitro.

Using a murine model of nematode infection, we have discovered macrophages that display a novel phenotype that may be characteristic of macrophages in chronic type 2 inflammation. These nematode-elicited macrophages (NeMphi) are characterized by two unique features: the ability to actively suppress proliferation of a broad range of cell types and the high level expression of two novel macrophage genes, Ym1 and Fizz1. NeMphi also show some similarities with in vitro-derived 'alternatively activated macrophages' such as the downregulation of inflammatory cytokines. We therefore investigated how much of the phenotype discovered in vivo could be replicated by activation with Th2 cytokines in vitro. Fizz1 and Ym1 were upregulated by IL-4 and IL-13 in vitro but at a considerably lower level than in NeMphi. In vitro treatment with IL-4 could also partly replicate the ability of NeMphi to block cellular proliferation. As well as the quantitative differences in gene expression and suppressive phenotype, we also observed phenotypic differences in the cell morphology between macrophages activated in vivo and in vitro. Although this study illustrated that macrophages activated in chronic inflammation have distinct features that cannot be readily reproduced in vitro it also demonstrated that some features of the complex NeMphi phenotype can be replicated by treatment of cultured macrophages with Th2 cytokines. In future, we hope to use in vitro analysis to help define the pathways that lead to this distinctive in vivo macrophage phenotype.

Animals↗
PubMed1998

A comparison of low volume, Draize and in vitro eye irritation test data. III. Surfactant-based formulations.

The third phase in a series of investigations of the relationship between low volume eye test (LVET) data, Draize eye irritation test data, and comparable data from in vitro assay protocols is presented. These investigations utilize Draize eye test and in vitro endpoint data generated previously as part of the CTFA Evaluation of Alternatives Program. LVET data were generated de novo using the same 25 representative surfactant-based personal-care formulations. In general, these formulations were minimally to moderately irritating. The linear correlation between maximum average score as determined by the Draize test (MAS) and the LVET (LVET-MAS) was 0.87; LVET-MAS values were typically about 30% lower then corresponding MAS values. Comparison of in vitro assay performance with that of the LVET was determined by statistical analysis of the relationship between LVET-MAS and in vitro endpoint. Regression modelling was the primary means of enabling such a comparison, the objective being to predict LVET-MAS for a given test material (and to place upper and lower prediction bounds on the range in which the LVET-MAS is anticipated to fall with high probability) based on observation of an in vitro score for that material. The degree of 95% confidence in prediction is quantified in terms of the relative widths of prediction intervals constructed about the fitted regression curves. Twenty in vitro endpoints were shown to have the greatest agreement with the LVET (these endpoints included those with low discordance rates relative to the Draize test) and were therefore selected for regression modelling. Although prediction interval widths tended to be narrower when predicting LVET-MAS compared with predicting MAS, the confidence with which the selected in vitro endpoints predicted both LVET-MAS and MAS for surfactant-based formulations was greatest when values were close to the lower or upper limits of the observed irritation range (i.e. 95% prediction interval widths were most narrow in these areas). Overall precision of LVET-MAS prediction for surfactant-based formulations was similar to that previously reported for hydroalcoholic formulations and considerably better than was reported for oil/water emulsions.

Alcohols↗
PubMed2002

Currently available in vitro methods used in the regulatory toxicology.

About two decades ago in vitro mutagenicity tests were adopted as the first in vitro methods in regulatory toxicology. For reasons of animal welfare and better science, many validation studies of various in vitro methods were performed between 1985 and 1995 for their potential to replace the Draize eye irritation test. Albeit the fact that four in vitro methods (HET-CAM, BCOP, IRE, and ECE) have gained regulatory tolerance in Europe for the classification of severe eye irritants, the lessons learned mainly from these validation studies led to the definition of internationally harmonised OECD validation principles and acceptance criteria (Final Report of the OECD Workshop on Harmonization of Validation and Acceptance Criteria for Alternative Toxicological Tests Methods, 1996. OECD Publication Office, Paris, France). Application of these principles to prevalidation and validation studies, and additional special studies performed in Europe with the support of ECVAM, ended in the scientific validation of new in vitro methods for the prediction of skin corrosivity and phototoxicity. They were accepted for regulatory use in Europe and adopted on 8 June 2000 as test methods B. 40 and B. 41 of Annex V of Directive 67/548/EEC. In quite a different approach, European industry has submitted in-house validation data to support a Draft OECD Test Guideline for in vitro dermal absorption testing, which-after peer review and a long lasting international discussion-will now be adopted by the OECD. The increasing importance of regulatory measures derived from quantitative cytotoxicity tests, and some examples of regulatory accepted in vitro methods, where the specific purpose is restricted to a specific regulation (e.g. testing of medicinal products, or medical devices), are also addressed.

Animal Testing Alternatives↗
PubMed1997

Comparison of loads on internal spinal fixation devices measured in vitro and in vivo.

The loads on internal spinal fixation devices were measured using modified, telemeterized AO-Dick internal fixators. The implants allow measurement of the three force components and the three moments acting on the implant. The modified fixators were mounted on cadaver spines, and the implant loads were measured in the intact and postcorpectomy spines for different loading modes, including axial compression force, flexion, extension, lateral bending, and torsion. The in vitro experiment did not consider muscle forces. Modified fixators were also implanted in three patients, and the implant loads were determined before and after anterior interbody fusion with autologous iliac-crest bone grafts. The results for different in vitro loading modes were compared with those in vivo in order to demonstrate the extent to which the in vitro loads represent the real situation in patients. In several cases, the implant loads in the in vitro experiment differed strongly from those measured in patients. For flexion and lateral bending, a tensile axial force occasionally was measured in the in vitro experiment, while in the patients the axial force was always compressive. Extension was predominantly associated with extension bending moments in the in vitro study but with flexion bending moments in the patients. When muscle forces are not considered in the in vitro experiment, the loads on the fixators may differ significantly from the situation found in patients.

Adult↗
PubMed1993

Vitrification of rat blastocysts developed in vitro.

Approximately 50% of rat 8-cell embryos obtained from the oviduct on day 4 of pregnancy developed to the blastocyst stage after 18 of incubation in vitro. The embryos were compared with in vivo blastocysts derived from the uterus on day 5 of pregnancy as regards their response to vitrification treatment. Before vitrification, both types of embryos were exposed to vitrification solution, and subsequent embryonic development was inhibited with increasing time of exposure. A greater suppression of development after exposure was observed in the embryos cultured in vitro compared with in vivo blastocysts. However, development was gradually restored as in vitro incubation was continued. The response of embryos to vitrification and warming treatments was shown to be almost the same for both in vitro and in vivo blastocysts, though the former were significantly (p < 0.05) less tolerant of exposure to vitrification solution. These results suggest that in vitro blastocysts were much more susceptible to vitrification solution than in vivo blastocysts. The degree of damage caused by vitrification and thawing treatments, and the inhibition of in vitro embryonic development, were significantly (p < 0.05) more serious for in vitro blastocysts.

Animals↗
PubMed1996

In vitro-in vivo myotoxicity of intramuscular liposomal formulations.

PURPOSE: The first objective was to study the in vitro myotoxicity of empty liposomes and to examine whether liposome size, charge and fluidity affect of liposomal myotoxicity. The second objective was to investigate the effect of liposomal encapsulation on the in vitro and in vivo myotoxicity of loxapine compared to the loxapine commercial preparation (Loxitane). METHODS: The in vitro myotoxicity of empty liposomes and loxapine liposomes was evaluated by the cumulative efflux of the cytosolic enzyme creatine kinase (CK) from the isolated rat extensor digitorum longus (EDL) muscle over a 2 hour period. In the in vivo studies, the area under plasma CK curve over 12 hours was used to evaluate muscle damage. RESULTS: The in vitro myotoxicity for all empty liposomal formulations was not statistically different from negative controls (untreated control muscles and normal saline injected muscles). However, these empty liposomal formulations were significantly less myotoxic than the positive controls (muscles injected with phenytoin and muscle sliced in half). In vitro-in-vivo studies showed that the liposomal encapsulation of loxapine resulted in significant (P < 0.05) reduction in myotoxicity (80% in vitro and 60% in vivo) compared to the commercially available formulation which contains propylene glycol (70% V/V) and polysorbate 80 (5% W/V) prepared at equal concentration. CONCLUSIONS: Results indicate that empty liposomes do not induce myotoxicity. Furthermore, liposomal size, charge and fluidity do not affect myotoxicity. In addition, in vitro and in vivo studies have demonstrated that liposomal encapsulation of loxapine can reduce myotoxicity compared to a formulation containing organic cosolvents.

Animals↗
PubMed1995

Increase in chloride-dependent L-glutamate transport activity in synaptic membrane after in vitro ischemic treatment.

The effect of energy failure on Cl(-)-dependent L-glutamate (L-Glu) transport was examined with an in vitro preparation. Rat brain slices were incubated in low oxygen and glucose-deprived medium (in vitro ischemia), and a synaptic membrane fraction was prepared from the slices. Cl(-)-dependent L-[3H]Glu uptake into vesicles increased about twofold after 20 min of in vitro ischemia. The increased L-[3H]Glu uptake was inhibited by L-Glu, DL-2-amino-4-phosphonobutyrate, L-homocysteic acid, L-cystine, 4,4'-diisothiocyano-2,2'-disulfonic stilbene, and removal of Cl-. Uptakes of Na(+)-dependent L-[3H]Glu, [3H]GABA, and [3H]taurine were not changed by the in vitro ischemia. In vitro ischemia increased the Vmax value without affecting the Km value. The increased L-[3H]Glu uptake by in vitro ischemia was reduced by subsequent incubation in a normoxic glucose-containing solution. ATP content in brain slices decreased to < 10% of control values by in vitro ischemia for 10 min. The decrease in ATP content was restored by subsequent incubation in normoxic glucose-containing solution. Treatment with veratrine, 2,4-dinitrophenol, carbonyl cyanide m-chlorophenylhydrazone, and NaCN in normoxic conditions increased L-[3H]Glu uptake with a concomitant decrease in ATP content in slices. These results suggest that Cl(-)-dependent L-Glu transport activity in synaptic membranes increases in ischemia- or hypoxia-induced brain energy failures.

ATP-Binding Cassette Transporters↗
PubMed2001

Fluvoxamine-theophylline interaction: gap between in vitro and in vivo inhibition constants toward cytochrome P4501A2.

OBJECTIVE: Several reports indicate that fluvoxamine decreases the clearance of cytochrome P4501A2 (CYP1A2) substrates. This study compared in vitro and in vivo inhibition potencies of fluvoxamine toward CYP1A2 with an approach based on inhibition constants (K(i)) determined in vitro and in vivo. METHODS: In vitro inhibition constant values were determined with human liver microsomes and complementary deoxyribonucleic acid-expressed CYP1A2 (supersomes). Fluvoxamine in vivo inhibition constants (K(i)iv) for CYP1A2 were obtained from an investigation of single-dose theophylline (250 mg) disposition in 9 healthy volunteers receiving steady-state (9 days) fluvoxamine at 3 doses (0, 25, or 75 mg/d) in a randomized crossover design. RESULTS: In vitro K(i) values based on total inhibitor concentrations were 177 +/- 56 nmol/L, 121 +/- 21 nmol/L, and 52 +/- 13 nmol/L in human liver microsomes with 1 mg/ml protein and 0.5 mg/ml protein and in supersomes with 0.3 mg/ml protein, respectively. The corresponding in vitro K(i) values based on unbound fluvoxamine concentrations were 35 nmol/L, 36 nmol/L, and 36 nmol/L. The ratio of 1-methyluric acid formation clearances (control/inhibited) in 8 subjects was positively correlated with fluvoxamine concentration (r (2) = 0.87; P <.001) with an intercept near 1. Mean values for K(i)iv based on total and unbound plasma concentrations at steady state were 25.3 nmol/L (range, 14-39 nmol/L) and 3.6 nmol/L (range, 2.4-5.9 nmol/L), respectively. CONCLUSION: Comparison of in vitro and in vivo K(i) values based on unbound fluvoxamine concentrations suggests that fluvoxamine inhibition potency is approximately 10 times greater in vivo than in vitro.

Adult↗
PubMed1998

Post-natal growth and development of Simmental calves derived from in vivo or in vitro embryos.

Large fetuses arising from embryos produced in vitro have been shown to exhibit altered organ development in utero, but it is not known whether this persists post natally. Post-natal growth and development was examined in 18 Simmental bulls derived from in vivo frozen-thawed (n = 6), in vitro frozen-thawed (n = 6) or in vitro fresh (n = 6) embryos and reared together post weaning on an ad libitum diet until slaughter at approximately 13 months old. Calves weighing less than 60 kg at birth (n = 11) were classified as normal, and heavier calves (n = 7; all from in vitro embryos) as oversize. Lifetime growth rates and slaughter weights apparently were unaffected by embryo source or birthweight. Mean (+/- s.e.m.) post mortem liver and kidney weights were unaffected by embryo source, but hearts of bulls from in vitro frozen embryos were heavier than those of bulls from in vivo frozen embryos (2.7 +/- 0.04 v 2.3 +/- 0.07 kg, P<0.025). Heart weight per kilogram body weight at slaughter for the 7 perinatally oversize males (4.01 +/- 0.08 g) exceeded that of the other 5 bulls from in vitro embryos (3.60 +/- 0.10 g kg(-1); P<0.04) and the 6 in vivo males (3.56 +/- 0.12 g kg(-1); P<0.02). Overall, one-third of the variation in heart weight at slaughter (r2 = 0.35; P = 0.01) was due to variation in birthweight. This is the first study to demonstrate birthweight-related developmental effects on post-natal organ weight following the transfer of embryos produced in vitro.

Animals↗
PubMed2004

Prediction of in vivo drug interactions with eplerenone in man from in vitro metabolic inhibition data.

1: The inhibition kinetics of eplerenone (EP) 6beta-hydroxylation by 10 drugs were determined in vitro using human liver microsomes. Inhibition factors were calculated from in vitro inhibition constant (Ki) and three different inhibitor Cmax values (liver Cmax of total and unbound inhibitor, and maximum influx concentration of inhibitor into the liver). Subsequently, the inhibition factors were compared with available pharmacokinetic data derived from clinical interaction trials conducted by Pfizer involving EP and these drugs. EP was also evaluated for its effect on the metabolism of 10 drugs in vitro, and again the in vitro data were compared with results from the clinical trials. 2: The Ki values for the inhibition of EP 6beta-hydroxylation by cisapride, cyclosporine, digoxin, erythromycin, fluconazole, ketoconazole, midazolam, saquinavir, simvastatin and verapamil were 2.90, 1.24,>75.0, 9.50, 59.0, 0.160, 8.10, 0.546, 6.23 and 13.3 microM, respectively. Among the three methods, inhibition factors (Rb) calculated using the Ki and estimated liver Cmax values of the unbound drug were best correlated with the in vivo area under the curve-fold increases of EP in humans. The Rb values for the drugs listed above were 1.04, 1.69, 1.00, 2.17, 2.24, 4.90, 1.00, 1.82, 1.01 and 1.04, respectively, and the in vivo area under the curve-fold increases of EP by these drugs were 1.04, 1.16, 0.930, 2.87, 2.24, 5.39, 1.00, 2.07, 1.03 and 1.98, respectively. 3: EP did not have any significant effects on the drugs tested in vitro or in the clinic. 4: Using in vitro metabolic interaction data, human in vivo pharmacokinetic interactions involving EP could be predicted nearly quantitatively. The lack of effects of EP on the pharmacokinetics of other drugs in man was also suggested in the in vitro data.

Drug Antagonism↗
PubMed2003

In vitro priming of myeloma antigen-specific allogeneic donor T cells with idiotype pulsed dendritic cells.

In vitro priming of T cells with dendritic cells (DC) pulsed with clinically relevant, but weak antigens such as tumor idiotype (Id), is an attractive strategy to generate tumor-specific T lymphocytes. In order to enhance the specific antitumor effect of allogeneic stem cell grafts, we investigated whether induction of tumor specific T cells using autologous DC pulsed with patient's myeloma Id could be maintained and potentiated by in vitro priming. For induction of T cells, DC (5 x 10(5)/well) were cultured with autologous nonadherent cells (DoNA) (5 x 10(6)/well) and antigen (TT10 microg/ml, KLH 100 microg/ml and Id 100 microg/ml). The T cells were restimulated every 8-10 days with the corresponding antigen and autologons DC. After 2-4 cycles of in vitro priming, the T cells were compared with nonadherent cells obtained after 2h attachment on day 0 (DoNA) for antigen-specific cytokine production. In vitro primed T cells (2-4 cycles of stimulation with Ag and DC) showed significant antigen-specific cytokine responses (IFN-gamma, TNF-alpha, GM-CSF) to TT. Similarly, in vitro priming of T cells to Id-pulsed DC resulted in marked increases in cytokine production for both myeloma Id proteins tested. These data suggest that multiple in vitro immunization using DC could be beneficial in generating tumor specific T cells from normal donor PBMC, which may be used for adoptive immunotherapy (e.g. "tumor-specific" donor lymphocyte infusion) of B cell malignancies. In vitro immunization may also offer an alternative to immunization of healthy stem cell transplant donors with tumor antigen.

Antigen Presentation↗
PubMed1981

A comparison of in vivo and in vitro methods for determining availability of iron from meals.

A comparison is made between in vitro and human and rat in vivo methods for estimating food iron availability. Complex meals formulated to replicate meals used by Cook and Monsen (Am J Clin Nutr 1976;29:859) in human iron availability trials were used in the comparison. The meals were prepared by substituting pork, fish, cheese, egg, liver, or chicken for beef in two basic test meals and were evaluated for iron availability using in vitro and rat in vivo methods. When the criterion for comparison was the ability to show statistically significant differences between iron availability in the various meals, there was substantial agreement between the in vitro and human in vivo methods. There was less agreement between the human in vivo and the rat in vivo and between the in vivo and the rat in vivo and between the in vitro and the rat in vivo methods. Correlation analysis indicated significant agreement between in vitro and human in vivo methods. Correlation between the rat in vivo and human in vivo methods were also significant but correlations between the in vitro and rat in vivo methods were less significant and, in some cases, not significant. The comparison supports the contention that the in vitro method allows a rapid, inexpensive, and accurate estimation of nonheme iron availability in complex meals.

Animals↗
PubMed2001

In vitro starch digestion correlates well with rate and extent of starch digestion in broiler chickens.

Current feed evaluation systems for poultry are based on digested components (fat, protein and nitrogen-free extracts). Digestible starch is the most important energy source in broiler chicken feeds and is part of the nitrogen-free extract fraction. Digestible starch may be predicted using an in vitro method that mimics digestive processes in the gastrointestinal tract of broiler chickens. An experiment was designed to use this method for predicting site, rate and extent of starch digestion in broiler chickens. In vitro starch digestion was studied in 12 experimental diets differing in starch sources. These diets were also used in a digestibility trial with broiler chickens. Correlations between in vitro and in vivo starch digestion were calculated. Starch digestion after 2 h incubation correlated well with in vivo starch digestion in the first half of the small intestine (r = 0.94). A 4-h incubation period resulted in a good correlation between in vitro starch digestion and ileal starch digestion (r = 0.96). In vitro starch digestion rate (h(-1)) correlated well with in vivo starch digestion rate (r = 0.87). In vitro starch digestion of individual starch sources was additive. It appeared that legume seeds and waxy corn contained two starch fractions, which were digested at different rates. We conclude that starch digestion rate in broiler chickens is well predicted by the in vitro method.

Animal Feed↗
PubMed2004

Evaluation of an in vitro dialysis system to predict drug removal.

BACKGROUND: Variation in the extent of drug removal under different dialysis conditions presents a challenge for prediction of drug elimination and dosage regimen adjustment during haemodialysis (HD). Dependence on clinical pharmacokinetic studies in HD patients for dosing guidelines is problematic given the increasing number of dialysers with variable rates of drug removal. Thus, the purpose of this study was to characterize drug removal using an in vitro system and to evaluate its reliability to predict in vivo elimination by HD using vancomycin (VANC) as a model drug. METHODS: In vitro dialysis was performed for 2 h (volume 4.0 l normal saline, initial VANC concentration 30 mg/l, flow rate 300 ml/min, dialysate flow 800 ml/min) using four different dialysers: polymethylmethacrylate (BK-2.1 U), polysulfone (F-80), AN69 (Filtral-20) and hemophan (COBE 700HE). The in vitro dialysis clearance for VANC (CL(D)) for the polysulfone dialyser was compared with values determined in eight HD patients. In vitro VANC CL(D) for all dialysers was compared with the clearance and KoA for B12 reported for each dialyser. RESULTS: In vitro VANC CL(D) values were 93+/-11 ml/min for the polymethylmethacrylate BK-2.1, 136+/-7 ml/min for the AN69, 65+/-9 ml/min for the hemophan COBE 700HE and 143+/-10 ml/min for the polysulfone F80. The CL(D) for the polysulfone F80 was not statistically different from the in vivo CL(D) of 135+/-18 ml/min (P = 0.48). In vitro VANC CL(D) correlated with the B12 CL(D) (r(2) = 0.77) and the B12 KoA (r(2) = 0.63) reported for each dialyser. CONCLUSION: VANC CL(D) in HD patients for the polysulfone dialyser was correctly predicted using the in vitro dialysis system. Use of this system may be superior to estimations of drug CL(D) based on dialyser information provided by the manufacturer for compounds of similar molecular weight.

Adult↗
PubMed1994

Embryonic human chorionic gonadotropin secretion and hatching: poor correlation with cleavage rate and morphological assessment during preimplantation development in vitro.

Of 593 bipronucleate eggs allowed to develop in vitro, 275 (46%) achieved the blastocyst stage and beyond, 124 (21%) initiated hatching, but only 49 (8%) fully hatched. About half of the pre-embryos (48%) which developed to these more advanced stages were incapable of secreting significant amounts (> 200 microIU) of cumulative human chorionic gonadotropin (HCG) up to day 14. HCG production does not appear to begin until the expanded stage and is independent of hatching. Assessing cleavage rate through successive stages and morphological grades up to the 8-cell stage had little bearing on the ability of a pre-embryo to hatch or secrete HCG. Progression through the stages of preimplantation development in vitro does not always appear to be accompanied by the necessary biochemical stages. If only 46% of pre-embryos with two pronuclei are capable of achieving the blastocyst stage, and of these only 52% are capable of secreting HCG, then it follows that only 24% of the original bipronucleate pre-embryos in vitro can be considered anatomically and biochemically competent. However, this is only applicable for pre-embryos not transferred or frozen, and is thus subject to a selection bias. Inability to detect HCG in vitro is not conclusive proof that a pre-embryo is developmentally incompetent. Similarly failure to hatch in vitro may not be taken as definitive evidence that hatching would have failed had fertilization and development been completed in vivo. Nevertheless, if pre-embryonic development in vitro is similar to that in vivo, this may be a contributory factor in the low pregnancy rates following in-vitro fertilization treatment.

Blastocyst↗
PubMed2003

Chronology of apoptosis in bovine embryos produced in vivo and in vitro.

The postimplantation developmental potential of embryos can be affected by various forms of cell death, such as apoptosis, at preimplantation stages. However, correct assessment of apoptosis is needed for adequate inference of the developmental significance of this process. This study is the first to investigate the independent chronological occurrence of apoptotic changes in nuclear morphology and DNA degradation (detected by the TUNEL reaction) and incidences of nuclei displaying these features at various preimplantation stages of bovine embryos produced both in vivo and in vitro. Different elements of apoptosis were observed at various developmental stages and appeared to be differentially affected by in vitro production. Nuclear condensation was observed from the 6-cell stage in vitro and the 8-cell stage in vivo, whereas the TUNEL reaction was first observed at the 6-cell stage in vitro and the 21-cell stage in vivo. Morphological signs of other forms of cell death were also observed in normally developing embryos produced both in vivo and in vitro. The onset of apoptosis seems to be developmentally regulated in a stage-specific manner, but discrete features of the apoptotic process may be differentially regulated and independently modulated by the mode of embryo production. Significant differences in indices of various apoptotic features were not evident between in vivo- and in vitro-produced embryos at the morula stage, but such differences could be observed at the blastocyst stage, where in vitro production was associated with a higher degree of apoptosis in the inner cell mass.

Animals↗