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PubMed1999

The current status of equine embryo transfer.

The use of embryo transfer in the horse has increased steadily over the past two decades. However, several unique biological features as well as technical problems have limited its widespread use in the horse as compared with that in the cattle industry. Factors that affect embryo recovery include the day of recovery, number of ovulations, age of the donor and the quality of sire's semen. Generally, embryo recoveries are performed 7 or 8 d after ovulation unless the embryos are to be frozen, in which case recovery is performed 6 d after ovulation. Most embryos are recovered from single-ovulating mares. Because there is no commercially available hormonal preparation for inducing multiple ovulation in the horse, equine pituitary extract has been used to increase the number of ovulations in treated mares, but FSH of ovine or porcine origin is relatively ineffective in inducing multiple ovulation in the mare. Factors shown to affect pregnancy rates after embryo transfer include method of transfer, synchrony of the donor and recipient, embryo quality, and management of the recipient. One of the major improvements in equine embryo transfer over the last several years is the ability to store embryos at 5 degrees C and thus ship them to a centralized station for transfer into recipient mares. Embryos are collected by practitioners on the farm, cooled to 5 degrees C in a passive cooling unit and shipped to an embryo transfer station without a major decrease in fertility. However, progress in developing techniques for freezing equine embryos has been slow. Currently, only small, Day-6 equine embryos can be frozen with reasonable success. Additional studies are needed to refine the techniques for freezing embryos collected from mares 7 or 8 d after ovulation. Demand for the development of assisted reproductive techniques in the horse has increased dramatically. Collection of equine oocytes by transvaginal, ultrasound-guided puncture and the transfer of these oocytes into recipients is now being used to produce pregnancies from donors that had previously been unable to provide embryos. In vitro fertilization, however, has been essentially unsuccessful in the horse. One alternative to in vitro fertilization that has shown promise is intracytoplasmic sperm injection. However, culture conditions for in vitro-produced embryos appear to be inadequate. The continued demand for assisted reproductive technology will likely result in the further development of techniques that are suitable for use in the horse.

Animals↗
PubMed1999

Estimates of pregnancy outcomes based on selection of bovine embryos produced in vivo or in vitro.

The objective of this study was to estimate the degree of variation among experienced evaluators selecting in vivo- or in vitro-produced embryos for transfer and to determine how this affects both the proportion of recipients becoming pregnant after transfer, and the number of embryo transfers required per pregnancy. Data from 6 experienced evaluators who graded Day 7 embryos produced either in vivo (n = 15) or in vitro (n = 15) were used to estimate these effects. The evaluators viewed video recorded images of the embryos and classified each embryo for stage of development and quality grade (1 = excellent, 2 = good, 3 = fair, 4 = degenerated and nontransferable). The statistical model considered transfer of embryos of the following individual or combined grades: Grade 1 only, Grade 2 only, Grade 3 only, Grades 1 and 2, Grades 2 and 3, and Grades 1, 2 and 3. Probabilities of pregnancy after embryo transfer were based on pregnancy rates of recipients at the facility of 1 of the 6 evaluators where the percentages of heifers pregnant after the transfer of Grade 1, 2 and 3 embryos, by embryo source, were 76, 65 and 54% (in vivo), and 59, 45 and 30% (in vitro). Within most grades, the proportion of embryos selected for transfer differed (P < 0.05) among the 6 evaluators. Although no significant differences (P > 0.10) were found among evaluators in the proportion of recipients pregnant after transfer within any embryo grade, there was substantial variation among evaluators in the proportion of recipients becoming pregnant, especially for embryos produced in vitro. Estimated percentages of heifers becoming pregnant for embryos classified as Grade 1, 2 or 3 were 66 to 76, 62 to 69, and 54 to 60%, respectively, for in vivo-produced embryos; and, 39 to 59, 15 to 45, and 24 to 32%, respectively, for in vitro-produced embryos. Approximately twice as many transfers were needed per pregnancy for embryos produced in vitro as for those produced in vivo regardless of the grade.

Animals↗
PubMed1997

The frequency and developmental capability of human embryos containing multinucleated blastomeres.

The frequency of multinucleated blastomeres (MNB) in 2- and 4-cell stage human embryos was recorded immediately before embryo transfer using a high-power inverted microscope. About 44% of patients (150/338) possessed embryos exhibiting MNB. The appearance of this nuclear abnormality was not correlated with maternal age. Overall, 15% of the otherwise good quality embryos (274/1885) that developed after monospermic fertilization contained several multinuclei (from two to seven) in at least one cell. Quite often MNB were found within all cells of the embryo (50% in 2-cell embryos). Blastomere multinucleation was significantly higher in 2-cell than 4-cell embryos (P <0.0001). This suggests that a considerable number of human embryos become abnormal during the first embryonic division. The embryos containing MNB were usually excluded for uterine transfers, with the exception of 19 cases when only such embryos could be replaced (6%; 19/338 patients). The results demonstrated that embryos with MNB may implant (4/19 cases; 21%) and they can lead to both spontaneous abortions and the successful birth of healthy infants (two cases). The fact that in the successful cases, 2-cell stage embryos with a mononucleated and a binucleated blastomere were transferred also suggests that due to the cell totipotency, development of a healthy baby is possible from one normal blastomere. Since multinucleation in early embryos may reflect gross chromosomal abnormalities or development of mosaic embryos, it is advisable not to replace embryos with MNB. Occasional transfers, however, can be considered because defective embryos may sometimes develop normally.

Adult↗
PubMed2002

Quality and age of companion felid embryos modulate enhanced development by group culture.

For some species, embryos cultured with conspecific companions may have enhanced in vitro development compared with singletons. The objective of this study was to determine the effect of quality and age of companion embryos on single felid embryos produced by in vitro maturation or in vitro fertilization. Test oocytes (intermediate quality) were inseminated and incubated alone or with 10 embryos derived from oocytes with a high, intermediate, or low glucose uptake. The effect of relative age of companion embryos on test embryo development was also examined by insemination and incubation of test oocytes alone or with 10 conspecific embryos that were older, younger, or the same age. Test embryos coincubated with better- or equal-quality companions had better development and more cells per embryo (mean +/- SEM number, 74.9 +/- 16.9 and 40.6 +/- 8.8, respectively, Day 7; P < 0.05) than test embryos coincubated with lesser-quality companions (5.1 +/- 1.4) or alone (8.4 +/- 3.7). Intermediate-quality embryos incubated with older companions had more cells per embryo (88.3 +/- 17.0; P < 0.01) than those incubated with synchronous (49.3 +/- 12.1) or younger (29.4 +/- 6.1) embryos. The cell number of solitary embryos (9.8 +/- 3.1) was less (P < 0.05) than that of every group of test embryos incubated with companions, regardless of age. In vitro development of solitary cat embryos is improved by culture with excellent-quality conspecific companions, particularly companions of an advanced age.

Animals↗
PubMed2002

[Effects of cleavage-stage biopsy on in vitro development of human embryos].

OBJECTIVE: To evaluate the effects of biopsy methods, biopsy timing and the number of cell removed on in vitro development of embryos. METHODS: One hundred and fifty four embryos of good morphology from in vitro fertilization patients were studied. Sixty-six embryos were allocated to the following three groups: chemical drilling biopsy group (26), mechanical drilling biopsy group (26) and control group (20). One cell was removed from the embryos of the two biopsy groups. The remaining 88 embryos were allocated to two groups: biopsy group (44) and control group (44). Two cells were removed from biopsy group by chemical drilling method. The stage of the embryo before biopsy, biopsy time, lysed blastomere, growth potential and hatching capacity of the biopsied embryos, total cell number at the blastocyst stage were recorded and evaluated. RESULTS: The mean time of biopsy in the chemical drilling group (231 +/- 20) seconds was significantly shorter than that in the mechanical drilling group (262 +/- 23) seconds (P < 0.01). The proportion of embryo developing to blastocyst stage was higher in chemical drilling group as compared with the mechanical group (65% versus 35%, P < 0.05). The total cell number at the blastocyst stage was fewer than those in the 7 to 8-cell embryo and >/= 9-cell embryo groups in the 6-cell embryo group (44 +/- 4 versus 49 +/- 5, 50 +/- 6; P < 0.05). At 9- to 10-cell stage, the proportion developing to the blastocyst stage was reduced in compacted embryos (20%) compared with control group (67%, P < 0.05) also more lysed blastomeres after biopsy were found in compacted embryos (50%) compared with uncompacted embryos (17%). The growth capacity to the blastocyst stage and the total cell number of the blastocyst were not different between one cell removal group and two cells removal group (P > 0.05). However, the proportion developing to the blastocyst stage was reduced after the removal of two cells from the 6-cell stage in comparison to the control (1/8 versus 5/8, P < 0.05). CONCLUSIONS: Compared with mechanical drilling biopsy, chemical drilling technique takes shorter timer and is safer. The suitable biopsy timing was >/= 7-cell stage before embryo compacting. The removal of two cells from >/= 7-cell would not impair in vitro development of embryos.

Adult↗
PubMed1997

Viability of partially damaged human embryos after cryopreservation.

In our centre, embryos are judged to have survived cryopreservation if at least half of the initial number of blastomeres remain intact. Therefore both fully intact and partially damaged embryos are transferred. The aim of this study was to investigate the viability of partially damaged human embryos after cryopreservation. We retrospectively analysed the implantation and in-vivo development of embryos which were either fully intact or had lost some blastomeres after cryopreservation. Oocytes were collected following stimulation with the gonadotrophin-releasing hormone (GnRH)-agonist Buserelin and human menopausal gonadotrophin. Supernumerary multicellular embryos with not more than 20% of their volume filled with anucleate fragments were frozen on day 2 or day 3 of the cycle using a slow cooling procedure with dimethylsulphoxide as the cryoprotectant. Following slow thawing, 431 fully intact embryos were transferred in 314 embryo transfer procedures and 488 partially damaged embryos were transferred in 327 such procedures. The percentage of gestational sacs with fetal heartbeat obtained after transfer of fully intact embryos was almost three times higher than that after transfer of partially damaged embryos (11.4 versus 3.5%). Forty-five children (birth rate 10% per embryo transfer) were born after transfer of fully intact embryos and 14 after transfer of embryos from which some blastomeres had been lost following cryopreservation. In conclusion, although children have been delivered after transfer of partially damaged embryos, the aim of a cryopreservation programme must be to obtain fully intact embryos after thawing.

Blastomeres↗
PubMed2003

Amplification of representative cDNA pools from single human oocytes and pronucleate embryos.

In the human embryo, gene expression studies have been hindered by the scarcity of material and the fact that in vitro fertilisation (IVF) embryos available for research are usually of poor quality and are, therefore, not representative of normal development. This has led most authors to study individual human embryos, using conventional RT-PCR strategies, which permit analysis of only a few genes. Variability in the expression of genes between individual embryos is characteristic of these studies. In this study, a global RT-PCR strategy has been used, allowing the analysis of an almost infinite number of genes from a single embryo. We have used oocytes, which failed to fertilise and representative pronucleate embryos donated from cycles in which the patient conceived, to investigate possible variability in transcript abundance between individual embryos. We have screened oocytes and embryos for a panel of genes including beta-actin (expressed in 24/28 oocytes, 6/6 pronuclear embryos), the integrins beta1 (17/28 oocytes, 6/6 pronuclear embryos) and beta5 (8/28 oocytes, 5/6 pronuclear embryos), and the apoptotic regulators BCL-2 (20/28 oocytes, 2/6 pronuclear embryos) and BAX (21/28 oocytes, 5/6 pronuclear embryos). The expression of the pro-apoptotic regulator BAX increased in human oocytes following prolonged periods of culture. Overall, patterns of gene transcript presence showed variation between embryos and this was independent of either zona removal or lysis conditions. Pronucleate embryos showed less variation, however, even sibling embryos from the patient did not express an identical subset of genes.

DNA, Complementary↗
PubMed2007

Heat stress-induced apoptosis in porcine in vitro fertilized and parthenogenetic preimplantation-stage embryos.

Decades worth of research have consistently shown the adverse effects of elevated temperatures on reproductive parameters of livestock species. The objective of this study was to evaluate the developmental and apoptotic responses of porcine in vitro fertilized (IVF) and parthenogenetically activated (PA) embryos heat stressed at the late 1-cell stage. Embryos were heat stressed (HS) at 42 degrees C for 9 hr starting 22 hr after insemination or artificial activation stimulus. Non heat-stressed (NHS) control embryos were maintained at 39 degrees C for the duration of the experiments. TUNEL staining on Day 5 of development demonstrated that heat stress elicited a significant apoptotic response in IVF embryos (45.6% of HS embryos and 26.7% of NHS embryos were apoptotic; P<0.05), but not in PA embryos (51.1% and 39.9% for HS and NHS embryos, respectively; P>0.1). And, while IVF embryos were highly susceptible to heat-induced developmental perturbations (20.6% and 8.8% development to blastocyst for NHS and HS embryos, respectively; P<0.05), elevated temperatures did not affect blastocyst rates in PA embryos (22.2% for NHS PA embryos and 21.2% for HS PA embryos; P>0.1). These findings indicate that, as in other systems studied, IVF pig embryos are directly affected adversely by heat stress conditions. Parthenogenetic embryos, though, appear to be surprisingly tolerant of the elevated temperatures. The differences between IVF and PA embryos in their response to heat stress warrants further investigation.

Animals↗
PubMed1991

Co-culture of day-5 to day-7 equine embryos in medium with oviductal tissue.

Oviductal and uterine embryos were collected from mares at 5 to 7 days following ovulation 1) to evaluate the effects of oviductal tissue explants on in vitro growth and development of equine embryos and 2) to study the morphologic development of equine embryos in culture. Embryos were incubated for 5 days in a medium (control group) or in medium supplemented with oviductal tissue explants (co-culture group). Embryos were evaluated and the media changed daily. Following 5 days in culture, 10 10 (100%) control embryos and 27 29 (93%) co-cultured embryos had doubled in diameter. All embryos that were recovered as morulae developed to the blastocyst stage in culture. By 5 days in culture, 6 10 (60%) control embryos and 19 29 (66%) co-cultured embryos had reached the hatching blastocyst stage of development. By 3 days in culture, significantly more (P<0.05) control embryos versus co-cultured embryos had degenerated (4 10 vs 2 29 , respectively). By 5 days in culture, significantly more (P<0.01) control embryos versus co-cultured embryos had degenerated (6 10 vs. 3 29 , respectively). Embryos cultured with oviductal tissue were sustained longer than embryos cultured in medium alone. Hatching was characterized by the blastocyst squeezing through a small opening in the zona pellucida or by the zona pellucida thinning over approximately half of the blastocyst surface and subsequently disappearing entirely.

Journal Article↗
PubMed1991

Analysis of cryoprotectant, cooling rate and in situ dilution using conventional freezing or vitrification for cryopreserving sheep embryos.

Two studies were conducted to evaluate the influence of cryoprotectant, cooling rate, container and cryopreservation procedure on the post-thaw viability of sheep embryos. In Study 1, late morula- to blastocyst-stage embryos were exposed to 1 of 10 cryoprotectant (1.5 M, glycerol vs propylene glycol)-plunge temperature treatments. Embryos were placed in glass ampules and cooled at 1 degrees C/min to -5 degrees C, seeded and further cooled at 0.3 degrees C/min to -15, -20, -25, -30 and -35 degrees C before rapid cooling by direct placement in liquid nitrogen (LN(2)). Post-thaw embryo viability was improved (P<0.01) when embryos were cooled to at least -30 degrees C before LN(2) plunging. Although there were no overt differences in embryo viability between cryoprotectant treatments (each resulted in live offspring after embryo transfer), there was a lower (P<0.01) incidence of zona pellucida damage using propylene glycol (4%) compared to glycerol (40%). In Study 2, embryos were equilibrated in 1.5 M propylene glycol or glycerol or a vitrification solution (VS3a). Embryos treated in propylene glycol or glycerol were divided into ampule or one-step((R)) straw treatments, cooled to -6 degrees C at 1 degrees C/min, seeded, cooled at 0.5 degrees C/min to -35 degrees C, held for 15 minutes and then transferred to LN(2). Embryos vitrified in the highly concentrated VS3a (6.5 M glycerol + 6% bovine serum albumin) were transferred from room air to LN(2) vapor, and then stored in LN(2). Propylene glycol- and glycerol-treated embryos in straws experienced lower (P<0.05) degeneration rates (27%) and yielded more (P<0.05) hatched blastocysts (73 and 60%, respectively) at 48 hours of culture and more (P<0.05) trophoblastic outgrowths (67 and 53%, respectively) after 1 week than vitrified embryos (47, 40 and 20%, respectively). In vitro development rate for VS3a-treated embryos was similar (P>0.10) to that of ampule controls, which had fewer (P<0.05) expanded blastocysts compared to similar straw treatments. Live offspring were produced from embryos cryopreserved by each straw treatment (propylene glycol, 3 of 7; glycerol, 1 of 7; VS3a, 2 of 7). In summary, freeze-preservation of sheep embryos was more effective in one-step straws than glass ampules and propylene glycol tended to be the optimum cryoprotectant. Furthermore, these findings demonstrate, for the first time, the biological competence of sheep embryos cryopreserved using the simple and rapid procedure of vitrification.

Journal Article↗
PubMed1991

Comparison of pregnancy rates from transfer of fresh versus cooled, transported equine embryos.

Donor mares of mixed, light-horse breeds, maintained at Colorado State University, provided 104 embryos for immediate transfer (fresh embryos). One hundred and thirty-six additional embryos were collected on various breeding farms in the United States and were shipped to Colorado State University via commercial airlines (cooled embryos). Embryos were harvested 7 d after ovulation, graded, and either transferred into a mare immediately (<1 h) or placed in Ham's F-10 medium plus 10% fetal calf serum in an atmosphere of 5% CO2, 5% O2, 90% N2 and packaged in a passive cooling unit (Equitainer) for shipment to our laboratory. All embryos were measured and graded just prior to surgical transfer via flank incision into synchronized mares. Recipients had ovulated 1 or 2 d before (+1, +2), on the same day as (0), or 1, 2 or 3 d after (-1, -2, -3) the donor mare. Pregnancy of recipients was determined by ultrasonography on 12, 35, and 50 d after ovulation of the donor. Pregnancy rates at 12, 35, and 50 d were similar for fresh (74, 64, 61%) and cooled embryos (80, 67, 66%), respectively. Overall, embryo size affected (P<0.05) pregnancy rates at 12, 35 and 50 d. Embryos of Grade 1 (excellent) or 2 resulted in more pregnancies than those of Grade 3 or 4 (poor) embryos. Embryonic losses between 12 and 35 d or between 35 and 50 d were not altered (P>0.05) by treatment (fresh or cooled) nor by age of the donor mare (P>0.05), but embryonic losses between 12 and 35 d were greater (P<0.06) for embryos stored for >12 h (25%) versus those stored for <12 h (10%). The duration needed for shipment (<12 h or >12 h) of cooled embryos did not alter pregnancy rates at 12 d (P>0.05). Age of donor mare had no effect (P>0.05) upon pregnancy rates of cooled or fresh embryos transferred nor on embryo quality. In summary, equine embryos can be cooled to 5 degrees C and maintained in storage for up to 24 h without decreased fertility, compared with those of embryos transferred in <1 hour.

Journal Article↗
PubMed1994

Effects of visible light and other environmental factors on the production of oxygen radicals by hamster embryos.

Previous studies have demonstrated that developing hamster embryos are very sensitive to visible light. In order to elucidate why visible light exerts a toxic effect on hamster embryos, we examined the effect of visible light on the production of hydrogen peroxide (H(2)O(2)) within individual embryos, using a fluorimetric method. In addition, we examined the H(2)O(2) generating capacity of other factors which are known to be related to the in vitro developmental capacity of hamster embryos. One-cell hamster embryos were cultured with 2',7'-dichlorodihydrofluorescin diacetate, and the fluorescence emissions of the H(2)O(2)-dependent oxidative product in the embryos were measured using an Olympus microscopic photometry system. When embryos were exposed to visible light (14,000 lux) for a specified period (0, 0.5, 1, 2 or 3 min) prior to measurement, the fluorescence emissions from embryos increased with the time of exposure to visible light. An exposure of even 0.5 min resulted in a significant increase in hydrogen peroxide. This increase was more rapid in embryos cultured under 20% O(2) than in those cultured under 5% O(2), and the response was quicker than that observed in mouse embryos. The fluorescence emissions from embryos cultured under 5% O(2) were significantly (P<0.001) lower than those from embryos cultured under 20% O(2) in TLP medium. However, the effects of different oxygen tensions on fluorescence emissions were medium-dependent, and were not significant in embryos cultured in HECM-1 medium. The addition of L-cysteine to or elimination of phenol red from the media decreased the fluorescence emissions from embryos (P<0.001), but glucose and phosphate did not affect them. These results suggest that the toxic effect of visible light on the in vitro development of hamster embryos might be due to increased generation of reactive oxygen species, induced by the visible light. This could be one of the explanations for the strict conditions required for overcoming the in vitro developmental block. It is also suggested that the promotive effects of low oxygen culture and L-cysteine on embryo development seem to be derived from their ability to reduce reactive oxygen species.

Journal Article↗
PubMed2001

The use of microangiography in detecting aberrant vasculature in zebrafish embryos exposed to cadmium.

Embryonic vascular patterns in zebrafish (Danio rerio) could be visualised by confocal microscopy coupled with microinjected fluorescent microbeads. This microangiographic technique was adopted here, for the first time, to study the effects of cadmium on cardiovascular development in zebrafish embryos. Zebrafish embryos were incubated in culture medium containing 100 microM cadmium from 5 h post fertilisation (hpf) to 48 hpf. At 48 hpf, embryos were examined for viability and occurrence of malformations. The 100 microM cadmium caused 32.21 +/- 3.65% mortality and 20.33 +/- 4.04% visible malformations in surviving embryos. In the remaining embryos with no visible signs of malformations, further assessments for less obvious abnormalities were performed. Assessments on craniofacial development were made by digital measurements on areas of brains and eyes. Cardiac development was assessed by immunostaining the heart with the antibody MF20 specific for myosin heavy chain. Body lengths of the embryos were also measured. Embryonic development of brains, eyes, hearts and body lengths of visibly healthy embryos in the cadmium treatment group showed no significant difference from the controls. Embryonic vasculature of these visibly healthy embryos was then studied by microinjecting fluorescent microbeads of diameter 0.02 microm into the circulation. All the cadmium treated embryos showed localised vascular defects in the dorsal aortae, segmental and cranial vessels while none of the control embryos showed any aberrant patterns in the networking of the vasculature. Improved image analyses on the anterior regions revealed that cadmium treated embryos had markedly less complex networks of cranial vessels with fewer vessels perfusing the craniofacial regions. The number of branch points in the vascular network was counted. In untreated embryos, there were 135.6 +/- 51 branches in the vasculature in entire body. In the cadmium treated embryos, there were 64.5+/-31 branches. The difference was significant when assessed with Student's t-test. It appeared that although cadmium did not cause any signs of external malformations in these visibly healthy embryos, nonetheless induced impaired branching and anastomsis of the cranial vessels. This study revealed, for the first time, that vital vascular structures in fish embryos could be affected by exposure to cadmium. This technique allowed visualisation of vascular anomalies in embryos showing no external signs of malformations. The impairment of anatomical features during embryonic development might serve as meaningful health endpoints in ecotoxicological studies and in risk assessment.

Angiography↗
PubMed1994

Developmental competence of domestic cat embryos fertilized in vivo versus in vitro.

Development of in vitro-fertilized (IVF) cat embryos was compared to that of naturally produced cat embryos in vivo and in vitro. To obtain in vivo-fertilized embryos, queens were mated three times daily on the second and third days of natural estrus and ovariohysterectomized at 64, 76, 100, 124, or 148 h after the first copulation. Embryos were flushed from the reproductive tract, evaluated for developmental stage, and cultured. For IVF, oocytes from gonadotropin-stimulated queens were inseminated with electroejaculated cat sperm in Ham's F-10 and evaluated for fertilization (cleavage to > or = 2 cells) at 30 h. In vitro development of embryos fertilized in vivo (n = 109) and in vitro (n = 46) was evaluated every 24 h for up to 10 days. High-quality embryos recovered at 64, 76, 100, 124, and 148 h after the first copulation were typically 1 to 2 cells (13 of 20), 5 to 8 cells (18 of 28), 9 to 16 cells (14 of 24), morulae (15 of 21), and compact morulae (11 of 18), respectively, suggesting blastomere cleavage once per day in vivo after the first three rapid cell divisions. A similar developmental rate to the morula stage (p > or = 0.05) was achieved in vitro by embryos derived from both in vitro and in vivo fertilization. Additionally, the proportion (p > or = 0.05) of in vivo-generated embryos (2 to 16 cells) that developed to morulae (64 of 83; 77.1%) was similar to that of IVF embryos (28 of 46; 60.9%). However, none of the IVF embryos (0/46), but 70.6% (77 of 109) of the in vivo-produced embryos, achieved blastocyst formation in culture (p < or = 0.05). Furthermore, 66.2% (51 of 77) of these blastocysts exhibited zona hatching. Incidence of morula and blastocyst formation in the in vivo group was influenced by stage of the embryo at collection. Embryos that were at the 9- to 16-cell stage at recovery were more likely (p < or = 0.05) to achieve morula or blastocyst status and emerge from the zona pellucida than younger-stage counterparts. In summary, the in vivo and in vitro growth rate of cat embryos produced after natural mating was comparable to that of embryos fertilized and cultured in vitro. However, developmental ability to the blastocyst stage was superior for embryos produced in vivo after natural mating.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1992

Effect of heat-stress on bovine embryo development in vitro.

Chronic elevation of uterine temperature has long been known to increase embryo mortality in dairy cattle. Short-term elevation in temperature of mouse embryos to 43 degrees C (acute) has been shown to induce intracellular production of heat-shock proteins. In this study, in vitro development of bovine embryos was assessed during short-term (60 h) coculture with oviduct epithelial cells at 38.6 degrees C (T1), 40 degrees C (T2), 38.6 degrees C after a prior pulse treatment (20 min) at 43 degrees C with 5% CO2 (T3), or 38.6 degrees C after a prior pulse treatment (20 min) at 43 degrees C with 100% CO2 (T4). During incubation, embryos cocultured at 40 degrees C had a greater (P < .05) mean embryo development score at 36 h than embryos cocultured at 38.6 degrees C. At 60 h of incubation, embryo development scores were greater (P < .05) for embryos cultured at 38.6 degrees C than for those cocultured at 40 degrees C. The number of embryos hatched at 60 h was similar after coculture at 38.6 degrees C (T1) or a prior pulse treatment with 5% CO2 and 43 degrees C (T3), but the embryo development score at 60 h was greater (P < .05) for the pulse-treated embryos. Embryos in T4 had greater (P < .05) embryo development scores than did T1 embryos from 36 through 60 h. Pulse treatment (T4) resulted in a greater (P < .05) number of hatched embryos at 60 h than T1, T2, and T3. These results indicate a detrimental effect of a chronic elevation in temperature that was evident shortly after embryo hatching.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Release of somatic embryogenic potential from excised zygotic embryos of carrot and maintenance of proembryonic cultures in hormone-free medium.

Excised zygotic embryos, mericarps ("seeds") and hypocotyls of seedlings of cultivated carrot Daucus carota cv. Scarlet Nantes were evaluated for their ability to generate somatic embryos on a semisolid hormone-free nutrient medium. Neither intact zygotic embryos nor hypocotyls ever produced somatic embryos. However, mericarps and broken zygotic embryos were excellent sources for somatic embryo production (response levels as high as 86%). Somatic embryo formation was highest from cotyledons, but was also observed on isolated hypocotyls and root tips of mature zygotic embryos. On media containing unreduced nitrogen, somatic embryo formation led to the generation of vigorous cultures comprised entirely of somatic embryos at various stages of development which in turn proliferated still other somatic embryos. However, a medium was devised which when 1-5 mM NH4+ was the sole nitrogen source, led only to a proliferation of globular proembryos. Sustained subculturing of these proembryos at 2-3 week intervals enabled establishment of highly uniform cultures in which no further development into more mature stages of embryonic development occurred. These have been maintained, without decline, as morphogenetically competent proembryonic globules for over ten months. A basal medium containing from 1-5 mM NH4+ as the sole nitrogen source appears not to be inductive to somatic proembryo formation. Instead, such a medium is best thought of as permissive to the expression of embryogenically determined cells within zygotic embryos. By excising and breaking or wounding zygotic embryos, constituent cells are probably released from positional or chemical restraints and thus are able to express their innate embryogenic potential. Once a proembryonic culture is established, this medium containing 1-5 mM NH4+ as the sole nitrogen source provides a nonpermissive environment to the development and growth of later embryonic stages, but it does allow the continued formation and multiplication of globular somatic proembryos. The sequence of events leading from excised broken zygotic embryos to the formation of somatic embryos and the maintenance of somatic proembryos are demonstrated by scanning electron microscopy and histological preparations. Germination levels from intact zygotic embryos on media with varying levels and ratios of unreduced vs. reduced inorganic nitrogen were determined as well and provided baseline or control data on the type of response obtained from nonwounded material.

Ammonium Chloride↗
PubMed1996

Radiosensitive target in the mouse embryo chimera assay: implications that the target involves autocrine growth factor function.

Mouse preimplantation embryos express at least two functional cell surface growth factor receptors that are radiosensitive in other cell types, the epidermal growth factor receptor (EGF receptor) and the insulin-like growth factor I receptor (IGF-I receptor). These embryos also express ligands that bind to and activate these receptors, including transforming growth factor alpha (TGF-alpha) and insulin-like growth factor II (IGF-II), which bind to the EGF receptor and IGF-I receptor, respectively. Embryo-expressed IGF-II and TGF-alpha increase embryo cell number--a measure of proliferation rate--and stimulate blastocoele formation--a measure of cell differentiation--allowing the embryo to self-modulate cell proliferation and morphogenesis into a blastocyst (Paria and Dey, Proc. Natl. Acad. Sci. USA 87, 4756-4760, 1990; Dardik and Schultz, Development 113, 919-930, 1991; Rappolee et al., Genes Dev. 6, 939-952, 1992). In this work, we tested the hypothesis that IGF-I receptor and/or EGF receptor function may be impaired to produce the radiation-induced competitive cell proliferation disadvantage that is expressed by irradiated embryos that are aggregated with nonirradiated embryos in chimeras. Cleavage-stage embryos were irradiated with 137Cs gamma rays (0.5 or 1.0 Gy) and paired with nonirradiated same-stage embryos to form groups of chimeras that were cultured in control medium or medium containing IGF-II, insulin, EGF or TGF-alpha. The cell proliferation disadvantage expressed by the irradiated embryos within chimeras was completely eliminated by IGF-II or insulin. In contrast to the rescue action of IGF-II or insulin in chimeras, neither EGF nor TGF-alpha could prevent the cell proliferation disadvantage exhibited by irradiated embryos paired with nonirradiated embryos in chimeras. For irradiated conventionally cultured zona-enclosed embryos, IGF-II and TGF-alpha did not increase mean embryo cell number significantly, although both IGF-II and TGF-alpha did increase blastocoele formation significantly. Collectively, these results support the following conclusions: (1) Ligands for the IGF-I receptor can rescue irradiated embryos from competitive cell proliferation disadvantage in chimeras, while ligands for the EGF receptor cannot; (2) IGF-I receptor function and EGF receptor function are affected differently by ionizing radiation with respect to competitive cell proliferation and are affected similarly by ionizing radiation with respect to blastocoele formation; (3) EGF receptor-dependent stimulation of competitive cell proliferation and cell differentiation are affected differently by ionizing radiation in preimplantation embryos.

Animals↗
PubMed1986

Cryopreservation of human embryos: progress on the clinical use of the technique in human in vitro fertilization.

Embryo cryopreservation has been studied at Monash University since 1981 and has been available to patients since mid-1983. Of approximately 1200 patients' cycles of in vitro fertilization (IVF), 445 have had excess embryos which they requested to be frozen. To date 205 patients have requested thawing of their embryos and 144 have had frozen-thawed embryos replaced in utero, resulting in 16 pregnancies. Four of these pregnancies aborted, four are ongoing, and eight deliveries have resulted, including one stillbirth at 26 weeks and one set of twins. Analysis of the data collected to date shows that patients with three or more embryos frozen have a significantly higher pregnancy rate than patients with one or two embryos frozen (23 versus 4%, respectively). Embryo viability, but not embryo survival, following freeze-thawing is related to the degree of embryonic fragmentation and the cell stage at freezing. Eight-cell embryos had a significantly higher viability than other cleavage stages. Those resulting in pregnancy tended to be the faster-dividing eight-cell embryos and were undamaged after freezing and thawing. However, when considering all cleavage stages, there was little effect of freezing damage on embryo viability, providing that at least 50% of the cell complement of embryos were intact and the zona pellucida was undamaged. Nor was there any marked effect of the age of embryos postinsemination. It is also possible that patients who were pregnant following the initial embryo replacement on the cycle of IVF treatment are more likely to conceive following replacement of their frozen-thawed embryos.

Cell Count↗