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PubMed2003

Adriamycin effects on the chick embryo.

Adriamycin is an anthracycline, anti-neoplastic drug with known teratogenic effects on foetal rats in what is known as the Adriamycin rat model (ARM). This includes conditions similar to those in newborn humans, known collectively as the VACTERL association. This comprises vertebral (V), anorectal (A), cardiac (C), tracheoesophageal (TE), renal (R) and limb (L) anomalies. We designed this study to test the hypothesis that the administration of Adriamycin to chick embryos would cause similar anomalies to those in the VACTERL association seen in the ARM. Fertilized Ross eggs received Adriamycin doses from 2-50 microg into the air sac and from 0.9-6 microg into the albumin. Administration varied from day 0-3 (D(0-3)) with D(0) being the first day of incubation. Control eggs received saline. Embryos were incubated at 38 degrees C and a relative humidity of 70%. Embryos were recovered on D(14), paraffin-embedded and transverse sections studied for morphological abnormalities. In the air sac group ( n=142), 71% of Adriamycin embryos survived versus 86% of controls (n=29). In the albumin group (n=121), 42% of Adriamycin embryos survived versus 55% of controls (n=69). No embryos demonstrated anomalies consistent with the VACTERL association. Ventral defects affected 1% of surviving Adriamycin embryos versus 4% of controls in the air sac group. In the albumin group, 19.8% of surviving Adriamycin embryos had ventral defects compared to 15.7% of surviving controls. Anophthalmia affected 1% of the surviving embryos in the Adriamycin air sac group and 2% of the Adriamycin albumin group. No controls developed anophthalmia. Exencephaly affected 2% of the survivors in the Adriamycin air sac group but none of the albumin group or controls. The administration of Adriamycin to chick embryos in comparable doses and times to those used in the ARM does not appear to produce comparable effects in relation to developmental anomalies, such as the VACTERL association. Despite examining different administration routes and mimicking the ARM, by giving Adriamycin to embryos at gastrulation, we were unable to re-create the anomalies seen in the ARM.

Abnormalities, Drug-Induced↗
PubMed1987

Protection of rat embryos in culture against the embryotoxicity of acrolein using exogenous glutathione.

The aldehyde acrolein is embryotoxic in vivo and in vitro. Since acrolein is reactive towards thiols, glutathione was evaluated for its protective effects against the in vitro embryotoxicity of acrolein. Day 10 rat embryos were cultured in the presence of acrolein and glutathione, either concurrently or sequentially, and evaluated for embryo deaths, malformations, growth retardation, and content of glutathione and protein. Acrolein, added alone at the initiation of the culture period, was embryolethal to 64 and 100% of the embryos at 120 and 160 microM respectively. At acrolein concentrations of 80 and 120 microM, 50 and 100%, respectively, of the surviving embryos were malformed. In addition, both of these concentrations of acrolein produced growth retardation manifested by significant decreases in the yolk sac diameter, crown-rump and head lengths, number of somites, and morphological score. Concurrent exposure to 100 or 500 microM glutathione markedly protected embryos against all of these effects. To study the mechanism of the protective effect of glutathione against the embryotoxicity of acrolein, the effects of sequential addition of acrolein and glutathione were determined. When rat embryos were cultured in the presence of acrolein for 2 hr prior to exposure to glutathione, even 500 microM glutathione could not offer any protection against the embryolethality, teratogenicity, and growth retardation induced by acrolein. However, a 6-hr preincubation with 500 microM glutathione, prior to exposure to acrolein (in the absence of exogenous glutathione), significantly decreased the incidence of embryo deaths at 160 microM acrolein and brought the number of deaths and malformations among embryos exposed to 120 microM acrolein down to a level not significantly different from control; unlike the embryos exposed concurrently to acrolein and glutathione, however, the sequential treatment with glutathione and acrolein did not protect against growth retardation. While there were some changes in the total glutathione and protein content of embryos and yolk sacs with acrolein exposure, none of the treatments had any overall effect on the glutathione concentration per mg protein. Thus, exogenous glutathione can protect against the in vitro embryotoxicity of acrolein. We propose that this protection is mediated in part by a direct interaction between glutathione and acrolein, added concurrently to the serum medium, and in part by an indirect effect on the embryo of glutathione added prior to acrolein.

Abnormalities, Drug-Induced↗
PubMed1985

7,12-Dimethylbenz[a]anthracene-DNA adduct formation in Wistar rat and Syrian hamster embryo cell cultures.

The binding of 7,12-dimethylbenz[a]anthracene (DMBA) to DNA was examined in Syrian hamster and Wistar rat embryo cell cultures exposed to DMBA for 5, 24, 48 and 72 h. The level of binding of DMBA to DNA was about twice as great in the hamster embryo cells as in the rat embryo cells at all times. Analysis of the DMBA-deoxyribonucleoside adducts by immobilized boronate chromatography demonstrated that the ratio of adducts with no cis vicinal hydroxyl groups to those containing cis vicinal hydroxyl groups was much greater in the rat embryo cells (from 2.2:1 to 2.9:1) than in the hamster embryo cells (from 1.3:1 to 1.6:1). The hamster embryo cells contained three major DMBADE-DNA adducts: based upon their chromatographic behavior and comparison with the three major DMBA-DNA adducts described by Dipple et al. in mouse embryo cell cultures (Biochemistry, 24 (1985) 2291), two were tentatively identified as resulting from the reaction of anti-DMBADE (the isomer of 1,2-epoxy-3,4-dihydroxy-1,2,3,4-tetrahydro-DMBA with the epoxide and benzylic hydroxyl on the opposite faces of the molecule) with deoxyguanosine and deoxyadenosine and one adduct resulted from reaction of syn-DMBADE (epoxide and benzylic hydroxyl on the same face of the molecule) with deoxyadenosine. The anti-DMBADE-deoxyguanosine, syn-DMBADE-deoxyadenosine, and anti-DMBADE-deoxyadenosine adducts were present in hamster embryo cell DNA in a ratio of 1.2:2:1. The Wistar rat embryo cell DNA contained a much larger proportion of the syn-DMBADE-deoxyadenosine adduct. The relative proportions of the three major DMBA-DNA adducts in Syrian hamster embryo cells were similar at all times, but the proportion of syn-DMBADE-deoxyadenosine adduct decreased slightly with time in the rat embryo cells. These results indicate that there are species specific differences in the stereospecificity of activation of DMBA to DNA-binding diol epoxides which parallel those observed for benzo[a]pyrene (BaP). The high proportion of deoxyadenosine adducts suggests that they may have an important role in the induction of biological effects by DMBA.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1983

Developmental autonomy of muscle fine structure in muscle lineage cells of ascidian embryos.

We have observed ultrastructural features of muscle differentiation in the muscle lineage cells of cleavage-arrested whole embryos and partial embryos of ascidians. Whole embryos of Ciona intestinalis and Ascidia ceratodes were cleavage-arrested with cytochalasin B at the 8-cell stage and reared to an age equivalent to several hours after hatching; these embryos formed extensive myofilaments which were often further organized into myofibrils of different sizes and densities in the peripheral cytoplasm of the two muscle lineage blastomeres (B4.1 pair). Developing myofibrils in cleavage-arrested embryos resembled the muscle elements observed in normal hatched larvae, but were less uniformly organized. A similar development of myofilaments and myofibrils occurred in the muscle lineage cells of multicellular partial embryos reared to "hatching" age. These partial embryos resulted from the isolated muscle lineage pair (B4.1) of blastomeres of the 8-cell stage (Ciona and Ascidia), and from a muscle lineage blastomere pair (B5.2) isolated at the 16-cell stage (Ascidia). Muscle lineage cells in the partial embryos were readily identified by the dense aggregates of mitochondria in their cytoplasm. Taken together, these results from the two kinds of partial embryo effectively eliminate inductive interactions with embryonic tissues other than mesodermal as a necessary factor in the onset of self-differentiation in muscle lineage cells. The relative complexity of muscle phenotype expressed in cleavage-arrested and partial embryos attests to an unusually strong developmental autonomy in the ascidian muscle lineages. This autonomy lends further support to the theory that a localized and segregated egg cytoplasmic determinant is responsible for larval muscle development in ascidian embryos.

Animals↗
PubMed1993

Arachidonic acid metabolism during early development of ovine embryos: a possible relationship to shedding of the zona pellucida.

Two experiments were conducted to determine if early ovine embryos produce prostaglandins and if prostaglandins may have a role in the shedding of the zona pellucida, i.e., embryo hatching. For Experiment 1, embryos were collected on day (d) 4, 8, 10, 12 or 14 of pregnancy and incubated with 1 microCi of [14C] arachidonic acid (AA) for 24 h. Based upon high-performance liquid chromatography, embryos from all days converted AA to a number of compounds; the amounts produced differed with day. Primarily, embryos produced an unidentified polar compound, 6-keto-PGF1 alpha, PGF2 alpha, PGE2, 13,14-dihydro-15-keto-PGF2 alpha and PGB2. For Experiment 2, embryos collected on d 7 of pregnancy were incubated for a maximum of 6 d in 500 microL of medium containing either ethanol (control; 20 microL), indomethacin (INDO; 10(-4) M), PGE2 (2 ng) or INDO (10(-4) M) + PGE2 (2 ng). Embryos were evaluated daily for hatching from the zona pellucida. The hatching rates (percentage) for control, INDO, PGE2 and INDO + PGE2 were 46.4, 34.5, 60.0, and 30.0, respectively. There was a main effect (P < .09) of treatment, and the hatching rate for embryos treated with PGE2 alone was greater (P < .05) than that for embryos in any group with INDO. The results indicate that early ovine embryos can convert AA to various compounds in vitro, and prostaglandins may have a role in the hatching of sheep embryos from the zona pellucida.

6-Ketoprostaglandin F1 alpha↗
PubMed1988

Effects of injecting antibodies to mouse nerve growth factor into the chick embryo.

The function of NGF in chick embryos was studied by injecting antibodies to mouse nerve growth factor (NGF). The uptake of mammalian antibodies into the 8- to 15-day-old chick embryo was followed by an enzyme-linked immunoassay. Normal rabbit antibodies (250 micrograms) were administered to the yolk, of which less than 5% was found in the embryo (300 ng of IgG per g wet wt of embryo). The concentration was proportionally lower when 100 micrograms anti-NGF antibodies were injected (40 ng/g). The concentration of anti-NGF antibodies was 1.5 times higher following injection directly into the body of the embryos. The effects of injecting antibodies at days 3-7 were studied in 10-day-old embryos by measuring the diameter frequencies of neurons in sympathetic and sensory ganglia. In comparison with controls, significantly smaller neurons were found in the sympathetic ganglia in embryos directly injected with anti-NGF. In the spinal ganglia, distribution of neuron diameters did not differ between anti-NGF-treated embryos and controls. Finally, the ability of different antibodies to mouse NGF to inhibit the in vitro activity of recombinant chick NGF was investigated. Total block was found at 1000-2000 ng of IgG per ml for most of the antibodies tested, levels not reached when injecting the embryo with antibodies to NGF. We conclude that the main reason for the limited effects in chick embryos by injection of NGF antibodies is due to the low levels of penetration of the anti-NGF IgG into the embryo.

Animals↗
PubMed1993

Increased generation of reactive oxygen species in embryos cultured in vitro.

Previous studies have suggested that oxygen toxicity is closely related to the developmental blockage of embryos cultured in vitro. In this study, to obtain an actual proof of the increase in production of reactive oxygen species within embryos, we have measured the level of H2O2 in individual embryos using a fluorimetric method. Mouse (ICR) pronuclear stage embryos from the oviducts were cultured for a specified time under various conditions in a medium to which 2',7'-dichlorodihydrofluorescin diacetate was added. After washing the embryos, the fluorescence emissions of the H2O2-dependent oxidative product in embryos were measured. The fluorescent emissions were lowest in embryos cultured under 5% O2 and highest under 40% O2 (5% < 20% < 40%), just the inverse of the culture efficacy. The fluorescence emmissions of embryos cultured in Ham's F-10, which contains hypoxanthine and transition metals such as Cu and Fe, were higher than those cultured in BWW and alpha MEM, which do not contain these components (alpha MEM < BWW < Ham's F-10; again this is the inverse of the culture efficacy). The fluorescence emissions of embryos increased with the time of the exposure to visible light. L-cysteine and thioredoxin, both of which have been shown to promote embryo development, decreased the fluorescence emissions of embryos. All of these results would provide direct evidence for the hypothesis that oxygen radicals are involved in the developmental blockage.

Animals↗
PubMed2006

Development and quality of sheep embryos cultured in commercial G1.3/G2.3 sequential media.

Present study assesses the developmental ability and quality of ovine IVP embryos derived from culture in sequential media G1.3/G2.3. A total of 1474 cumulus-oocyte complexes were matured in M199 supplemented with EGF and FCS for 24h in 5% CO2 in humidified air at 39 degrees C. Oocytes were co-incubated in SOF medium with 1 x 10(6) spermatozoa/ml at the same temperature and gas conditions (Day 0 p.i.). Presumptive zygotes at 20 h p.i. were denuded, washed and placed in culture in SOF (control; n=742) or G1.3 media supplemented with 3mg/ml of BSA (n=732) under mineral oil in a humidified and controlled atmosphere at 39 degrees C. Embryos in the treated group were changed to G2.3 medium on Day 3 of culture. A group of blastocysts in each group were frozen by conventional method (SOF, n=55; G1.3/G2.3, n=48). In vivo embryos (n=72) were recovered at Day 7 from the uterus of progestagen+eCG treated females and they were cultured in defined medium (n=38) or frozen (n=34) directly after recovery. Cleavage rate of IVP embryos recorded at 48 h p.i. was similar for control and treated embryos (49.8 versus 47.5%). There were no significant differences in blastocyst development from the two groups on Day 6 (26.0 versus 25.6%), 7 (42.1 versus 38.6%) or 8 (50.8 versus 43.2%). Blastocyst development rates from total oocytes cultured were comparable (24.1 versus 21.5%). However, the proportion of hatched blastocysts was significantly higher for control embryos (86.6 versus 44.3%, P<0.0001). In addition, embryos cultured in SOF had higher re-expansion rates post-thawing at 24h (38.2 versus 6.2%), 48 h (36.4 versus 4.1%) and 72 h (34.5 versus 4.1%) and hatching rate (32.8 versus 2.0%) than embryos cultured in sequential media (P<0.0001). In vivo embryos showed higher hatching rate (61.7%) than IVP groups (SOF, P<0.01; G1.3/G2.3, P<0.0001) but lower than their fresh cultured counterparts (86.8%; P=0.01). In conclusion, G1.3/G2.3 media supported high developmental rates of embryos in vitro but the quality of the embryos was impaired.

Animals↗
PubMed2004

The anterior-posterior axis emerges respecting the morphology of the mouse embryo that changes and aligns with the uterus before gastrulation.

BACKGROUND: When the anterior-posterior axis of the mouse embryo becomes explicit at gastrulation, it is almost perpendicular to the long uterine axis. This led to the belief that the uterus could play a key role in positioning this future body axis. RESULTS: Here, we demonstrate that when the anterior-posterior axis first emerges it does not respect the axes of the uterus but, rather, the morphology of the embryo. Unexpectedly, the emerging anterior-posterior axis is initially aligned not with the long, but the short axis of the embryo. Then whether the embryo develops in vitro or in utero, the anterior-posterior axis becomes aligned with the long axis of embryo just prior to gastrulation. Of three mechanisms that could account for this apparent shift in anterior-posterior axis orientation-cell migration, spatial change of gene expression, or change in embryo shape-lineage tracing studies favor a shape change accompanied by restriction of the expression domain of anterior markers. This property of the embryo must be modulated by interactions with the uterus as ultimately the anterior-posterior and long axes of the embryo align with the left-right uterine axis. CONCLUSIONS: The emerging anterior-posterior axis relates to embryo morphology rather than that of the uterus. The apparent shift in its orientation to align with the long embryonic axis and with the uterus is associated with a change in embryo shape and a refinement of anterior gene expression pattern. This suggests an interdependence between anterior-posterior gene expression, the shape of the embryo, and the uterus.

Animals↗
PubMed2004

Blastomere transplantation in human embryos may be a treatment for single gene diseases.

OBJECTIVE: To determine whether human embryos accept blastomere transplants and integrate them normally into the architecture of the developing embryo. DESIGN: A human blastomere transplantation model, involving 44 cryopreserved embryos that were specifically donated to research. SETTING: Academically affiliated private infertility center. PATIENT(S): Forty-four human embryos. INTERVENTION(S): In 21 experiments, one, two, or three blastomeres were transplanted, using standard microsurgical techniques that are widely used in preimplantation genetic diagnosis (PGD). Embryos were thawed and gender was determined, using established PGD techniques. Male (xy) blastomeres were then transplanted into female (xx) day 3 embryos, and the xy cells were tracked through blastocyst stage (days 5-6) and into the hatching period (day 6), using fluorescent in situ hybridization (FISH). MAIN OUTCOME MEASURE(S): Degree and location of xy cell integration into xx embryos. RESULT(S): High-quality recipient embryos (with 4 to 10 cells) developed uniformly into normal blastocyst stage embryos in 12 of 12 experiments (100%) and integrated donor blastomeres into their architecture, with apparently even distribution of daughter cells; this integration was documented in inner cell mass as well as in trophoectoderm. The intensity of this distribution appeared to correlate with the number of blastomeres transferred. Among nine abnormally developing embryos, only three (33%) demonstrated a normal distribution of offspring donor cells. CONCLUSION(S): High-quality embryos appear to have the ability to integrate donor blastomeres. Because the treatment of single gene diseases does not require successful treatment of all cells, blastomere transplantation could be explored as a treatment option, which also would greatly enhance efficiency and utilization of preimplantation genetic diagnosis.

Blastocyst↗
PubMed2004

Noninvasive polarized light microscopy quantitatively distinguishes the multilaminar structure of the zona pellucida of living human eggs and embryos.

OBJECTIVE: To characterize the architecture of the zona pellucida in living human eggs and embryos, noninvasively with the PolScope, a digital polarizing light microscope. DESIGN: The PolScope was used to examine zonae pellucida of living human eggs and embryos. SETTING: Academic IVF clinic. PATIENT(S): Patients undergoing fresh, nondonor infertility treatment who underwent egg aspiration, fertilization by intracytoplasmic sperm injection or traditional IVF, and cleavage-stage embryo transfer (day 3). INTERVENTION(S): The PolScope imaged the zona of eggs before intracytoplasmic sperm injection and in cleavage-stage embryos before transfer. MAIN OUTCOME MEASURE(S): Thickness and retardance of three zona layers were measured from eight quadrants. Mean and variance in thickness and retardance were calculated for individual eggs and embryos, between eggs and embryos of a cohort, and across the sample patient population. RESULT(S): Cleavage-stage (day 3) embryos have thinner zonae (15.2 +/- 2.9 microm) than both immature (20.4 +/- 2.4 microm) and mature (19.5 +/- 2.2 microm) eggs. The zona of embryos is thinner, primarily owing to thinning of the outer layer. The thicker the zona layer, the greater its retardance. Considerable variation exists in the thickness and retardance of zona layers around individual eggs and embryos and between members of a cohort. The zona of eggs and embryos from different patients differ in thickness, retardance, and variation. CONCLUSION(S): Thickness and organization of zonae pellucida of human eggs and embryos varies considerably and can be quantitatively imaged with the PolScope.

Cleavage Stage, Ovum↗
PubMed2005

Spontaneous embryonic loss rates in twin and singleton pregnancies after transfer of top- versus intermediate-quality embryos.

OBJECTIVE: To determine whether embryo quality is associated with early spontaneous loss rates in twin and singleton pregnancies after IVF/intracytoplasmic sperm injection (ICSI). DESIGN: Retrospective, single center analysis. SETTING: The Center of Reproductive Medicine, Arcispedale Santa Maria Nuova, Reggio Emilia, Italy. PATIENT(S): Women undergoing IVF/ICSI and two- or three-embryo transfer of intermediate- and top-quality embryos. INTERVENTION(S): First-trimester sonography at 6 to 7 weeks to determine number of embryos with positive heartbeat. Number of embryos lost was calculated from a second-trimester sonogram. MAIN OUTCOME MEASURE(S): Rates of total pregnancy loss, as related to embryo quality, initial number of embryos, maternal age <35 or > or =35 years, and IVF procedure. RESULTS: A total of 94 losses (23.1% of 407 pregnancies) were counted, with similar proportions in pregnancies after transfer of intermediate- or top-quality embryos. Neither the mode of IVF procedure nor the number of transferred embryos affected the loss rate. In contrast, the loss rate was significantly higher in older mothers after transfer of intermediate-quality embryos (odds ratio [OR 2.4], 95% confidence interval [CI] 1.1-5.5). Losses among singletons were significantly higher compared with losses among twins (OR 2.5, 95% CI 1.1-6.0), but this was observed in top-quality embryos only. CONCLUSION(S): Top-quality but not intermediate-quality ETs are associated with lower early spontaneous loss rates among twin pregnancies after IVF/ICSI.

Abortion, Spontaneous↗
PubMed2006

In vitro blastocyst development from serially split mouse embryos and future implications for human assisted reproductive technologies.

OBJECTIVE: To assess the efficacy of serial splitting of mouse embryos with respect to blastocyst development. DESIGN: Prospective study. SETTING: Commercial research facility. ANIMAL(S): Commercially available mouse embryos from B6C3F-1 x B6D2F-1. INTERVENTION(S): One, two, and three blastomeres were biopsied from two-, four-, and six-cell embryos, respectively, and were inserted into empty zona pellucida recipients (first split). These embryos were cultured to reach their original cell number status and then were split again (second split). Once these embryos regained their original cell status, they were split yet again (third split). MAIN OUTCOME MEASURE(S): Blastocyst development of embryos split serially at the two-, four-, and six-cell stages. RESULT(S): The blastocyst development rate for two-, four-, and six-cell embryos subjected to a first split was 74.3%, 75.0%, and 66.6%, respectively, as compared with 71.8%, 62.6%, and 48.4% (second split) and 48.4%, 38.1%, and 10.6% (third split). CONCLUSION(S): First and second splitting of cleavage-stage embryos has yielded high efficiency rates for blastocyst development when compared with the third splitting, which did not provide any beneficial advantage for further embryo splitting and multiplication. This is the first study reporting on three serial embryo splittings in a mammalian species. Embryo splitting may have significant impact and applications in human assisted reproductive technology.

Animals↗
PubMed2004

Infectivity of bovine viral diarrhea virus associated with in vivo-derived bovine embryos.

Early research indicated that bovine viral diarrhea virus (BVDV) would not adhere to zona pellucida-intact (ZP-I), in vivo-derived bovine embryos. However, in a recent study, viral association of BVDV and in vivo-derived embryos was demonstrated. These findings raised questions regarding the infectivity of the embryo-associated virus. The objectives of this study were to evaluate the infectivity of BVDV associated with in vivo-derived bovine embryos through utilization of primary cultures of uterine tubal cells (UTC) as an in vitro model of the uterine environment and to determine if washing procedures, including trypsin treatment, were adequate to remove virus from in vivo-derived embryos. One hundred and nine ZP-I morulae and blastocysts (MB) and 77 non-fertile and degenerated (NFD) ova were collected on day 7 from 34, BVDV-negative, superovulated cows. After collection, all MB and NFD ova were washed according to International Embryo Transfer Society (IETS) standards and exposed for 2h to approximately 10(6) cell culture infective doses (50% endpoint) per milliliter of viral strain SD-1. Following exposure, some groups of <10 MB or NFD ova were washed in accordance with IETS standards. In addition, an equivalent number of MB and NFD ova were subjected to IETS standards for trypsin treatment. Subsequently, NFD ova were immediately sonicated and sonicate fluids were assayed for presence of virus, while individual and groups of MB were placed in microdrops containing primary cultures of UTCs and incubated. After 3 days, embryos, media, and UTCs were harvested from each microdrop and assayed for BVDV. Virus was detected in the sonicate fluids of 56 and 43% of the groups of NFD ova that were washed and trypsin-treated, respectively. After 3 days of microdrop culture, virus was not detected in media or sonicate fluids from any individual or groups of MB, regardless of treatment. However, virus was detected in a proportion of UTC that were co-cultured with washed groups of MB (30%), washed individual MB (9%) and trypsin treated individual MB (9%), but no virus was detected in the UTC associated with groups of trypsin-treated embryos. In conclusion, virus associated with developing embryos was infective for permissive cells. Further, the quantity of virus associated with a proportion of individual embryos (both washed and trypsin treated) was sufficient to infect the UTC. In light of these results, an attempt should be made to determine if the quantity of a high-affinity isolate of BVDV associated with an individual embryo would infect recipients via the intrauterine route.

Animals↗
PubMed2004

Rate of abnormalities in lambs from in vitro produced embryos transferred on Day 2 compared with Day 6 postfertilization.

The effect of transferring ovine IVP embryos on Day 2 versus Day 6 postinsemination was investigated. Oocytes were collected from 35 cull ewes and cultured separately for each donor. Embryos were exposed to serum in the maturation and fertilization media, and then cultured in a serum-free SOF system under serum-conditioned silicone oil. Cleaved Day 2 postfertilization embryos were randomly allocated for immediate transfer versus Day 6 transfer. Parturition was induced (with 15 mg dexamethasone) between Days 141 and 143 of pregnancy; those failing to initiate parturition by Day 146 had a Caesarean section. Stillborn lambs and those dying within several days of birth were necropsied. Lambs were weighed at birth; lambs (n = 132) derived from the same flock (from natural breeding or AI) were used as a control. An estimate of the 99th percentile of the normal population was used as the point above which lambs from three litter-size categories were classified as abnormally large (analysis was repeated for 95% confidence and median estimates of this value). Thirty-eight lambs were born from early transfer, 45 from late transfer and 12 from unobserved lambings (parentage could not be determined). Lambs from early transfer were heavier at birth (P = 6.0 x 10 (-11)). The early treatment group had fewer lambs produced per embryo transferred (P = 0.0002), fewer live lambs per embryo transferred (P = 0.00009) and fewer normal lambs per embryo transferred (P= 0.0002). There was an effect of donor on the number of dead lambs per embryo transferred (P = 0.017). The number of dystocias per transfer was not significantly different for early versus late transfer groups. However, the probabilities of survival for cleaved embryos were 27.3% and 13.1% in the early and late transfer treatment, respectively. There were more lambs produced per cleaved embryo with early transfer (P = 0.004) and there was an effect of donor within ram and replicate (P = 0.04) on the number of lambs produced. The odds ratio for early/late treatment was 2.449 with a confidence interval of 1.368-4.382. Day 2 transfer of cleaved embryos did not prevent the production of oversized or abnormal offspring; however, there was an increased chance of a cleaved embryo producing a lamb when transferred on Day 2 rather than Day 6 postfertilization.

Animals↗
PubMed2004

Sensitivity to chilling of medaka (Oryzias latipes) embryos at various developmental stages.

As an essential step toward cryopreservation of fish embryos, we examined the chilling sensitivity of medaka (Oryzias latipes) embryos at various developmental stages. Embryos at the 2-4 cell, 8-16 cell, morula, blastula, and early gastrula stages were suspended in Hanks solution. They were chilled to various temperatures (usually 0 degrees C), kept for various periods (usually 20 min), then cultured for up to 14 d to determine survival (assessed by the ability to hatch). Embryos at the 2-4 cell stage were the most sensitive to chilling to 0 degrees C, but sensitivity decreased as development proceeded. The survival rate of 2-4 cell embryos was affected after 2 min of chilling at 0 degrees C; although the rate decreased gradually as the duration of chilling increased, 38% of them still survived after 40 min of chilling. Embryos at the 2-4 cell stage were sensitive to chilling at 0 or -5 degrees C, but much less sensitive at 5 or 10 degrees C. The survival rate of 2-4 cell embryos subjected to repeated rapid cooling and warming was similar to that of those kept chilled. When early gastrula embryos were preserved at 0 or 5 degrees C, the hatching rate did not decrease after 12 and 24h of chilling, respectively, but then decreased gradually as storage was prolonged; however, 3-10% of the embryos hatched even after storage for 10 d. In conclusion, although later-stage medaka embryos would be suitable for cryopreservation (from the perspective of chilling sensitivity), chilling injury may not be serious in earlier stage embryos.

Animals↗
PubMed2006

Integrated morphophysiological assessment of two methods for sperm selection in bovine embryo production in vitro.

Extensive work was done regarding the ability of Swim up and Percoll gradient to select functional sperm for in vitro embryo production (IVP) systems. The aim of this work was to compare Swim up and Percoll as methods of sperm selection by ultrastructural, biochemical and functional studies. Frozen-thawed semen from two bulls (Experiments 1 and 2, respectively) were treated using Swim up or Percoll discontinuous gradients. Motility, sperm membrane ultrastructure, sperm proteins, in vitro embryo production (insemination doses, cleavage, embryo yield and quality) and embryo sex ratio were scored and compared. Electron transmission microscopy of outer sperm membranes showed higher (P<0.05) percentage of sperm with lost acrosomes in Percoll treated samples compared to Swim up. A differential protein pattern was also detected. When in vitro embryo production was performed, Percoll gradient produced higher (P<0.05) number of fertilizing doses (7.6 versus 5.9, Bull 1; 13.5 versus 7.8, Bull 2) and higher sperm motility (90% versus 76.6%, Bull 1; 81.7% versus 68.3%, Bull 2) than Swim up. The percentage of cleavage (Day 3) was similar in both treatment groups, whereas embryo production rate (Day 7) was higher (39.4% versus 30.2%, Bull 1; 38% versus 32.4%, Bull 2; P<0.05) when Percoll gradient was used. The percentage of hatched embryos (Day 11) and sex ratio did not differ. Total cell counting and embryo differential staining (inner cell mass and trophoblast cells) of Day 7 embryos showed that Percoll treated sperm produced better quality embryos compared to Swim up. We concluded that Percoll had a better performance selecting sperm and an enhanced capacity for embryo production when compared with the Swim up procedure; this could be attributed to a better acrosome exocytosis, associated to the absence of certain membrane proteins.

Acrosome↗
PubMed1988

Cryopreservation of zygotes and early cleaved human embryos.

Zygotes and 2- to 5-cell human embryos were frozen in 1,2-propanediol and sucrose; results of the first 50 cycles (45 patients) are presented. A total of 41 zygotes (17 attempts at thawing) were thawed, resulting in six singleton clinical pregnancies (15% per embryo; 35% per cycle), of which three delivered, one aborted, and two are ongoing. Fifty-seven cleaved embryos were thawed in 33 other cycles, resulting in four singleton and one twin pregnancy (11% per embryo; 15% per cycle), of which four delivered and one is ongoing. Depending on the cell stage, 61% to 81% of embryos survived cryostorage, but 2-cell embryos did not implant. One fifth of cryoinjury was due to the formation of cracks in the zona pellucida. The incidence of implantation was not enhanced when more than one freeze/thawed embryo was replaced, most pregnancies being obtained from single embryo replacements. At least 8% more births are expected in addition to conventional in vitro fertilization methods when the current policy of replacing three fresh embryos and freezing the remainder using this technique is applied. This method will result in two to four times more pregnancies per spare embryo, compared with other cryopreservation methods using older embryos.

Blastocyst↗