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PubMed2003

Incidence of chromosomal mosaicism in human embryos at different developmental stages analyzed by fluorescence in situ hybridization.

Chromosomal mosaicism has been reported in in vitro-cultured embryos at early cleavage stages, as well as in morulae and blastocysts. We have assessed the incidence and pattern of mosaicism during in vitro development of human embryos from early-cleavage stages to morula and blastocyst. Fifty spare embryos were fixed for fluorescence in situ hybridization (FISH) analysis for chromosomes X, Y, 13, 18, and 21 on days 2 or 3 (4- to 10-cell stage) (n = 16), on day 4 (morula stage) (n = 14), on day 5 (pre-expanded blastocyst) (n = 5), and the expanded blastocyst stages (n = 15). Blocked embryos (no cleavage observed within the last 24 hr) were not included. A total of 2367 cells were analyzed. Four early-cleavage stage embryos were found uniformly diploid; all of the others were mosaic for the chromosomes analyzed (mean diploid nuclei 48.3% +/- 28.7). All of the embryos at more advanced developmental stages, except one fully normal morula, had mosaic chromosome constitutions, with an increase in the percentage of diploid cells in morulae, pre-expanded, and expanded blastocysts, respectively (mean diploid nuclei 78.6% +/- 11.7, 66.0% +/- 20.8, 79.6% +/- 12.8), in comparison with earlier stages. Hypotheses about the origin of mosaicism and embryo regulation mechanisms will be discussed.

Aneuploidy↗
PubMed1991

Morphology and size of embryos recovered from superovulated cows.

A total of 691 normal embryos were recovered from 183 superovulated donor cows on the 5th and 6th days after the first insemination, and were examined for their morphology and size in relation to their developmental stage. There was no significant difference in the thickness of the zona pellucida, the diameter of the cell mass, and the overall diameter of the embryos among zygotes, 2-, 4-, 8- and 16-cell embryos, and morulae. In the blastocyst stage, however, the diameter of the cell mass and the overall embryo diameter were significantly greater and the zona pellucida was significantly thinner than in the earlier-stage embryos. The volume of the blastomere significantly decreased from zygote to morula in proportion to the increase in the number of blastomeres. The volume of the cell mass of 2-cell embryos was decreased by about 30% compared with that of zygotes and no increase in the volume of the cell mass was observed during the progression from 2-cell stage to morula. The diameter of the cell mass and the overall diameter of morulae recovered on the 6th day after the first insemination were significantly greater than those of morulae recovered on the 5th day.

Animals↗
PubMed1995

Early embryonic development in vitro and embryo transfer in the cat.

Ten female cats were given a total dose of 200 IU PMSG over 3 days to induce superovulation. One to four-cell stage embryos were collected by flushing the oviducts 48 to 54 hr after the initial 250 IU dose of hCG. Some of the normal embryos collected were examined for culture in Medium-199 supplemented with 20% FCS. After 72 hr of culture, 222/248 (89.5%) had developed to the morula stage, and by 96-168 hr, 110 (64.7%) out of 170 morulae had developed into blastocysts. Four to 12 embryos cultured in vitro per cat were transferred to one of the uterine horns of 12 recipients in which synchronous ovulation had been induced with hCG. All 4 recipients of embryos which had developed to the morula stage on culture day 3, 3 of the 5 recipients of blastocysts on culture days 4-6, and none of the 3 recipients of blastocysts on culture day 7 became pregnant. It is concluded that early feline embryos are capable of efficiently developing into transferable morulae in vitro by ordinary culture methods, but that there is partial developmental arrest from the morula to the blastocyst stage.

Animals↗
PubMed1988

Glucose metabolism by preimplantation pig embryos.

Pig embryos were collected, 2-7 days after oestrus, in modified BMOC-2 containing glucose as the only energy source. Embryos were incubated individually in medium containing [5-(3)H]-, [1-(14)C]- or [6-(14)C]glucose. Total glucose metabolism, as measured by [5-(3)H]glucose use, increased steadily from the 1-cell to the 8-cell stage. Total glucose use increased (P less than 0.05) at the compacted morula stage and was highest (P less than 0.05) at the blastocyst stage. Production of 14CO2 from embryos metabolizing [1-(14)C]glucose increased steadily from the unfertilized ovum to the 8-cell stage. Metabolism of [1-(14)C]glucose increased at the compacted morula stage (P less than 0.05) and continued to increase (P less than 0.05) to the blastocyst stage. Metabolism of [6-(14)C]glucose increased steadily from the unfertilized ovum to the compacted morula stage. Metabolism of [6-(14)C]glucose was highest (P less than 0.05) for the blastocyst stage. Percentage pentose phosphate pathway activity of total glucose metabolism before the 4-cell stage was higher (greater than 5%) than that of 8-cell to blastocyst stage embryos (approximately 1%). When embryo metabolism was determined on a per cell basis for each isotope, the compacted morulae stage (16 cells) had a higher total glucose metabolism than all other embryo stages (P less than 0.05), while early blastocyst (32 cells) and blastocyst (64 cells) stage embryos metabolized more [5-(3)H]glucose than all stages except compacted morulae (P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Ratio of inner cell mass and trophoblastic cells in demi- and intact pig embryos.

Pig morulae, early blastocysts and blastocysts were microsurgically bisected to produce zona-free demi-embryos or remained nonbisected with or without zona pellucida, and the presence of inner cell mass cells was determined using a differential fluorochrome staining technique. After 24 h of in vitro culture, all demi-embryos were classified into three categories, based on morphological criteria: 1, excellent; 2, fair; and 3, degenerated. The average number of total cells and inner cell mass cells in intact embryos cultured without zona pellucida for 24 h was higher (P < 0.05) than that for those with zona pellucida in morulae and early blastocysts. The percentage of demi-embryos without inner cell mass cells in these different morphological categories was 18.7%, 22.2% and 29.8% for morulae, respectively; 3.8%, 16.7% and 30.8% for early blastocysts, respectively; and 3.7%, 32.0% and 36.4% for blastocysts, respectively. The percentage of demi-embryos without inner cell mass cells was lower (P < 0.01) in demi-embryos classified in category 1 compared with category 3 in early blastocysts and in category 1 compared with categories 2 and 3 in blastocysts. Significant differences in the total number of cells and the number of inner cell mass cells were apparent among the three morphological categories of demi-embryos derived from morulae, early blastocysts and blastocysts. The ratio of total cells to inner cell mass cells was similar among intact pig embryos and the different morphological categories of demi-embryos derived from morulae, early blastocysts and blastocysts, with the exception of that between demi-blastocysts of category 1 and the other groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1998

Expression of RNA from developmentally important genes in preimplantation bovine embryos produced in TCM supplemented with BSA.

This study investigated the effects of a semi-defined culture system on the temporal pattern of expression of RNA from genes involved in compaction and cavitation: gap junction protein connexin43 (Cx43), desmosomal glycoproteins desmoglein 1 (Dg 1), desmocollins I, II and III (Dc I, Dc II, Dc III), desmosomal protein plakophilin (Plako); metabolism glucosetransporter-1 (Glut-1); RNA processing poly(A)polymerase (PolyA); heat shock protein 70.1 (HSP); and trophoblastic function trophoblast protein (TP) in bovine oocytes and embryos generated in vitro using TCM199 supplemented with BSA as the culture medium. Morulae and blastocysts derived in vivo were collected from superovulated heifers and also used for this study. Poly(A)+ RNA was extracted from pools of 20-50 oocytes or embryos, analysed by reverse transcription-polymerase chain reaction and the amplified fragments were verified by sequencing. Assays were repeated at least three times for each developmental stage and provided consistent results in all replicates. In bovine embryos produced in vitro, mRNA encoding Cx43 was detectable up to the morula stage, whereas blastocysts and hatched blastocysts did not express this gene. No transcripts were found for Dg 1 and Dc I throughout the tested preimplantation stages. Dc II and Dc III transcripts were found from 2-4-cell embryos up to the hatched blastocyst stage. mRNA encoding Plako was detected in immature and mature oocytes and zygotes, while no transcripts were seen in 2-4-cell and 8-16-cell embryos. The gene was expressed again from the morulae to the hatched blastocyst stage. Oocytes and bovine embryos produced in vitro showed transcripts for Glut-1, PolyA and HSP throughout preimplantation development up to the hatched blastocyst stage. The gene encoding TP was transcribed only in blastocysts and hatched blastocysts. Morulae and blastocysts produced in vivo showed the same expression as their in vitro counterparts, with one exception: the in vivo embryos transcribed Cx43. The results of this study reveal for the first time the transcriptional pattern of a set of 'marker' genes involved in various processes in early bovine embryonic development. Transferable morulae and blastocysts produced in vitro expressed most genes similar to their in vivo counterparts. These data contribute to the molecular characterization of this widely used in vitro culture system for bovine embryos and provide a major advance towards production of 'physiologically normal' embryos.

Animals↗
PubMed1999

Cloning of bovine embryos from vitrified donor blastomeres.

The use of cryopreserved in vitro produced bovine embryos as nuclear transfer donors was assessed. Day 4 or 5 morulae were vitrified and warmed using the open pulled straw method and used as donors for nuclear transfer. Although the proportion of morulae and blastocysts that developed from nuclear transfer embryos derived from day 5 vitrified embryos did not differ from that derived from fresh embryos (16.7 and 24.3%, respectively), development to blastocysts was reduced when vitrified donor cells were used (8.3 and 19.1%, respectively). Likewise, development to morulae and blastocysts was not different between nuclear transfer embryos derived from day 4 vitrified embryos allowed to recover for 24 h, and day 5 vitrified embryos allowed to recover for 1-2 h (27.7 and 15.6%, respectively), but the development to blastocysts was reduced when day 5 vitrified donor cells were used (23.2 and 10.0%, respectively). However, in nuclear transfer embryos derived from either day 4 vitrified or day 5 fresh donors, no differences were observed in development rates to morulae and blastocysts (34.3 and 36.3%, respectively) or to blastocysts alone (20.2 and 18.1%, respectively). Nor were there differences in the development rates of fresh or day 4 or day 5 vitrified in vitro produced (non-nuclear transfer) embryos (47.9, 51.0 and 35.5% developing to blastocysts at day 7, respectively). In vitro produced embryos and nuclear transfer embryos derived from day 4 vitrified or day 5 fresh donors were transferred to recipients at morula or blastocyst stage at day 6 or 7. The pregnancy rates were similar in both groups of nuclear transfer embryos, but higher in the control group consisting of in vitro produced embryos (47, 42 and 67%, respectively). In conclusion, if vitrified donor embryos are allowed to recover for 24 h after warming, their use in nuclear transfer results in similar efficiencies to those achieved with fresh embryos.

Animals↗
PubMed2005

Vitrification of biopsied mouse embryos.

Solid surface vitrification (SSV) was compared with in-straw vitrification for cryopreservation of biopsied mouse embryos. Eight-cell stage embryos were zona drilled and one blastomere was removed. Developed morulae or blastocysts were vitrified in microdrop (35% EG + 5% PVP + 0.4 M trehalose) or in straw (7.0 M EG + 0.5 M sucrose). Following recovery, embryos were cultivated in vitro or transferred into recipients. Cryopreservation had an effect not only on the survival of biopsied embryos but also on their subsequent development in vitro. Cryosurvival of biopsied morulae vitrified in straw was significantly inferior to SSV. The post-warm development of biopsied and non-biopsied morulae was delayed on Day 3.5 and 4.5 in both vitrification groups. A delay in development was observed on Day 5.5 among vitrified non-biopsied blastocysts. The percentage of pups born from biopsied morulae or blastocysts following cryopreservation did not differ from that of the control. No significant differences could be detected between methods within and between embryonic stages in terms of birth rate. The birth rate of biopsied embryos vitrified in straw was significantly lower compared to the non-biopsied embryos. The novel cryopreservation protocol of SSV proved to be effective for cryopreservation of morula- and blastocyst-stage biopsied embryos.

Animals↗
PubMed1993

Transfer of split goat embryos without zonae pellucidae either fresh or after freezing.

Embryos in the morula to blastocyst stage were collected from superovulated goats either surgically or after slaughter. Embryos with good or fair morphology were bisected with a microblade without the aid of a holding pipette or other microinstruments. Of 103 morulae and 77 blastocysts that were split, 200 (97%) and 151 (98%) demi-embryos, respectively, with no major morphological aberrations were obtained. Zona-free demi-embryos derived from blastocysts were incubated in vitro for 2 h, and those derived from morulae for 24 h so that they would reach the blastocyst stage. Demi-embryos were transferred either fresh or after freezing and thawing. After 2 h of incubation, a significantly higher proportion of zona-free demi-embryos derived from blastocysts were of good quality than were zona-free demi- embryos derived from morulae (75 vs 45%). Seventy-six percent of zona-free demi-embryos derived from morulae developed to the blastocyst stage after 24 h in vitro. The quality of the embryos before splitting had a significant effect on the development of the demi-embryos in vitro. Transfer of 11 pairs of zona-free demi-embryos resulted in the birth of five twin and three singleton kids. Transfer of 11 pairs of demi-embryos frozen-thawed without zonae pellucidae resulted in the delivery of two singleton kids.

Animals↗
PubMed2005

Effect of tumour necrosis factor-alpha (TNF-alpha) on protein synthesis by mouse preimplantation stage embryos in vitro.

Successful blastocyst implantation depends upon the synchronous dialogue between age- and stage-matched embryo and adequately primed maternal endometrium. Endometrial signals present in the uterine lumen influence the growth and the viability of preimplantation stage embryo. Thus, uterine secretion of embryotoxic cytokines may affect the preimplantation stage embryo. Our previous study in the rhesus monkey has indicated that tumor necrosis factor-alpha (TNF-alpha) is one such candidate present in the uterine lumen, which may act as an embryotoxic agent. In the present study, the effect of TNF-alpha on de novo protein synthesis by mouse morulae (n = 100) and blastocysts (n = 100) in vitro was investigated by 2D-polyacrylamide gel electrophoresis. A total of 35 and 40 protein spots were detected in lysates of control morulae and blastocysts, respectively. Exposure of embryos to TNF-alpha (50 ng/ml) reduced the number of protein spots to 15 and 17 compared to that of control morulae and blastocysts. Seven spots in morula and 13 protein spots in blastocyst flourograms showed quantitative changes in their expressions with exposure to TNF-alpha. Morulae and blastocysts exposed to TNF-alpha expressed 8 and 17 protein spots, respectively, that were not seen in control embryos. It appears from the present study that exposure of preimplantation stage embryos to TNF-alpha affects their protein synthesis both quantitatively and qualitatively.

Animals↗
PubMed1997

Nuclear transfer of porcine embryos using cryopreserved delipated blastomeres as donor nuclei.

Nuclear transfer protocol for the pig using cryopreserved delipated four- to eight-cell and morula stage embryos as nucleus donors was developed. Donor embryos, which had been delipated by micromanipulation following centrifugation for polarizing cytoplasmic lipid droplets, were cryopreserved with 1.5 M 1,2-propanediol and 0.1 M sucrose. Recipient cytoplasts were prepared from ovulated oocytes. Activation of oocytes could be induced more efficiently when electric stimulation was given 53 hr after the hCG injection or later (66-83%), compared with 52 hr or earlier (11-16%, P < 0.05), suggesting that aging after ovulation may be required for in vivo matured porcine oocytes to be activated by electric stimuli. Membrane fusion rates between donor blastomeres and enucleated oocytes were 88% (127/144) and 97% (56/58, P > 0.05) for the four- to eight-cell and morula stage embryos, respectively. In vitro developmental rates to the two-cell (53/100 vs. 35/65), four-cell (34/100 vs. 26/65), and morula stage (17/100 vs. 18/65) were the same between the nuclear transfer embryos with four- to eight-cell and morula nuclei. However, more embryos reconstituted with morula nuclei developed to blastocysts (15% vs. 6%, P < 0.05). These data demonstrated that blastomeres of cryopreserved, delipated porcine embryos can be used as donor nuclei for nuclear transfer. Frozen-thawed, delipated blastomeres can be efficiently isolated and fused, and therefore provide a useful source of donor nuclei.

Analysis of Variance↗
PubMed1991

Culture medium and protein supplementation influence in vitro fertilization and embryo development in the domestic cat.

The influence of culture medium and protein supplements on in vitro fertilization (IVF) and morula-to-blastocyst development in culture was examined in the domestic cat. In Study I, follicular oocytes were fertilized and cultured in 1) modified Krebs Ringer bicarbonate (mKRB); 2) modified Tyrode's solution (TALP) without phosphate or glucose; or 3) Ham's F10. All media contained bovine serum albumin (BSA). Fertilization rates were similar (P greater than .05) among mKRB (75.0%), TALP (70.6%), and Ham's F10 (80.0%) treatments. Compared to TALP (77.8%), more (P less than .05) embryos in Ham's F10 (95.0%) developed to the morula stage; development of mKRB embryos (88.9%) was intermediate and not different (P greater than .05). Study II evaluated the effects of protein availability and type on IVF and embryo growth. Ham's F10 was supplemented with polyvinylalcohol (PVA, 2 mg/ml), BSA (4 mg/ml), fetal calf serum (FCS, 5%), or estrous cat serum (ECS, 5%) and used to fertilize and culture embryos. Fertilization was enhanced (P less than .05) using either FCS (84.0%) or ECS (85.2%) compared to PVA (67.3%). Oocytes exposed to BSA fertilized at a rate (76.1%) similar (P greater than .05) to other treatments. Embryos exposed to all four treatment groups (PVA, 82.8%; BSA, 82.8%; FCS, 92.9%; or ECS, 97.8%) were equally capable (P greater than .05) of becoming morulae. However, more FCS- or ECS-supplemented morulae continued to the early blastocyst stage (30.8%, 22.2%, respectively, P less than .05) than PVA- (10.3%) or BSA- (13.8%) exposed morulae. In the domestic cat IVF system, the type of medium and protein supplement used appears to have a greater impact on embryo development in vitro than on fertilization.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Influence of the mucin coat on the survival of rabbit and mouse embryos stored at 0 degrees C.

To examine the factors affecting the survival of refrigerated embryos, rabbit and mouse morulae were stored at 0 degrees C in modified phosphate-buffered saline (PB1) or in PB1 containing 0.75 M sucrose. Survival was defined as the ability to develop into an expanded blastocyst in culture. The data was analyzed with special reference to the presence of a mucin coat around the embryos. When rabbit morulae were stored in isotonic PB1 for 2, 4, 6, and 8 days, survival rates were 98%, 88%, 85%, and 50%, respectively. However, if the mucin coat had been removed before storage, the rates were lower (95%, 75%, 36%, and 3%, respectively). Sucrose impaired the survival of rabbit morulae irrespective of the presence of the mucin coat. Only 11% of mouse morulae survived 2 days of storage in PB1 medium, but if the medium contained sucrose, survival rates after storage for 2, 3, 4, and 5 days were higher (83%, 55%, 31%, and 7%, respectively). To provide them with a mucin coat around the zona pellucida, mouse embryos were incubated in a rabbit oviduct. Survival rates of these embryos after storage in the presence of sucrose did not decrease over 4 days of refrigeration (98-92%), and the rates after storage for 5, 6, and 7 days were 65%, 40%, and 30%, respectively; embryos that had been stored for 5 days were transferred to recipient mice, and live young were born. Agar embedding of mouse morulae did not have the same effect as the mucin coat.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2002

Accumulation of cytoplasmic lipid droplets in bovine embryos and cryotolerance of embryos developed in different culture systems using serum-free or serum-containing media.

In this study, the quantitative fluctuation of cytoplasmic lipid droplets (LD) and cryotolerance were investigated in bovine embryos derived from in vitro-matured (IVM) and in vitro-fertilized (IVF) oocytes developed in different culture systems using serum-free or serum-containing media. The serum-free cultures were grown using IVMD101 medium in conjunction with bovine cumulus/granulosa cell (BCGC) cocultures or IVD101 medium without BCGC cocultures, and the serum-containing cultures were grown in the presence of BCGC cocultures using HPM199 medium supplemented with 5% calf serum (HPM199 + CS). Large numbers of sudanophilic LD were present in the cytoplasm of bovine embryos from 2-cell to hatched blastocyst stages, and the number and size differed between the embryos cultured in serum-free and serum-supplemented media. In the embryos cultured in HPM199 + CS, large (2-6 microm in diameter) sudanophilic LD increased significantly from the morula to the blastocyst stages. Throughout the embryonic development, the embryos developed in serum-free cultures with and without BCGC cocultures had numerous sudanophilic LD, but most of these droplets were small (<2 microm in diameter) and large LD were less numerous than those embryos cultured in HPM199 + CS. Giant LD (>6 microm in diameter) were frequently observed in morulae and blastocysts (including early blastocysts) developed in HPM199 + CS. Electron microscopic observations demonstrated that large LD were abundant in the cytoplasm of trophoblast and embryonic (inner cell mass) cells of blastocysts cultured in HPM199 + CS. These large LD were identified as osmophilic LD, an indication that these lipid inclusions contained a significant proportion of unsaturated lipids. Many elongated mitochondria were found in embryos developed in IVMD101 and IVD101 at the morula and early blastocyst stages, whereas many of the mitochondria in the morulae developed in HPM199 + CS were of an immature form such as spherical or ovoid shape. The survival and hatching rates of embryos (morulae, early blastocysts, and blastocysts) produced in serum-free media (both IVMD101 and IVD101) after post-thaw culture were superior to those of embryos produced in serum-containing medium. These results showed that bovine embryos cultured in serum-containing medium abnormally accumulated cytoplasmic lipids into their cytoplasm and the excess accumulation of cytoplasmic LD in embryos may affect the cryotolerance of embryos.

Animals↗
PubMed1998

Response of preimplantation murine embryos to heat shock as modified by developmental stage and glutathione status.

Objectives were to characterize developmental changes in response to heat shock in the preimplantation mouse embryo and to evaluate whether ability to synthesize glutathione is important for thermal resistance in mouse embryos. Heat shock (41 degrees C for 1 or 2 h) was most effective at disrupting development to the blastocyst stage when applied to embryos at the 2-cell stage that were delayed in development. Effects of heat shock on ability of embryos to undergo hatching were similar for 2-cell, 4-cell, and morula stage embryos. The phenomenon of induced thermotolerance, for which exposure to a mild heat shock increases resistance to a more severe heat shock, depended upon stage of development and whether embryos developed in vitro or in vivo. In particular, induced thermotolerance was observed for morulae derived from development in vivo but not for 2-cell embryos or morulae that developed in culture. Administration of buthionine sulfoximine to inhibit glutathione synthesis did not increase thermal sensitivity of 2-cell embryos or morulae but did reduce subsequent development of 2-cell embryos at both 37 degrees and 41 degrees C. In summary, changes in the ability of 2-cell through morula stages to continue to develop following a single heat shock were generally minimal. However, 2-cell embryos delayed in development had reduced thermal resistance, and therefore, maternal heat stress may be more likely to cause mortality of embryos that are already compromised in development. There were also developmental changes in the capacity of embryos to undergo induced thermotolerance. Glutathione synthesis was important for development of embryos but inhibition of glutathione synthesis did not make embryos more susceptible to heat shock.

Adaptation, Physiological↗
PubMed1992

Pregnancies and live birth from in vitro fertilization of calf oocytes collected by laparoscopic follicular aspiration.

Oocytes were recovered by laparoscopic aspiration from 3- to 8-week-old calves treated with follicle-stimulating hormone (FSH) followed by human chorionic gonadotropin (hCG) to induce follicular growth and oocyte maturation in vivo. Most of the recovered oocytes either had resumed meiotic maturation at the time of aspiration or were competent to undergo maturation during subsequent culture in vitro. Oocytes matured in vivo following FSH and hCG treatment underwent in vitro fertilization (70%) at rates not significantly different from those of control oocytes recovered from adult cow ovaries at abattoirs and matured in vitro (75%). Calf oocytes that were immature at aspiration exhibited lower fertilization rates after in vitro maturation (36%) but their rate of development to morulae and blastocysts did not differ from that of mature oocytes at aspiration. A total of 91% of the zygotes produced from calf oocytes developed to morula and 27% to blastocyst stages during 6 days of culture. The proportion developing to morulae was significantly higher (P<0.05) than that observed for zygotes resulting from in vitro maturation and fertilization of oocytes recovered from cow ovaries obtained at an abattoir and processed concomitantly (59% to morulae and 18% to blastocysts). Morulae or blastocysts developed from oocytes from 5 to 6-week-old calves, when transferred to synchronized recipient heifers, resulted in 2 confirmed pregnancies, one of which produced a single full-term live calf. The ability to produce embryos from oocytes recovered from newborn or prepubertal calves offers the potential for markedly reducing the generation interval in cattle, thereby substantially accelerating the rate of genetic gain that can be achieved through embryo transfer.

Journal Article↗
PubMed1995

Pronuclear visibility, development and transgene expression in IVM/IVF-derived porcine embryos.

A total of 1550 zygotes was used to assess the timing of pronuclear visibility, embryo development following DNA microinjection, and transgene expression in IVM/IVF-generated porcine embryos. After centrifugation, pronuclei could be seen in 61.6% of zygotes. In 55.3% of these only 1 pronucleus was visible. Pronuclear visibility was highest at 20 h post-insemination. Zygotes were microinjected with 1 of 2 LacZ gene constructs driven by either the SV40 early promoter (pSVON) or the human cytoplasmic beta actin promoter (pbActinLacZ). Development and transgene expression were assessed after either 48 h or 7 d in culture. After 48 h, significantly more zygotes with a single visible pronucleus developed to the 8-cell stage than zygotes in which no pronucleus had been seen (43.0 vs 24.8%), while those with 2 pronuclei were intermediate (31.4%). After 7 d, no difference in development to the morula stage was observed between noninjected control embryos (25.5%) and embryos with 1 (21.0%) or 2 pronuclei (22.5%); however, the proportion of embryos reaching the morula stage in the nonpronuclear group was significantly reduced (9.1%). After 48 h in culture, transgene expression was significantly higher in embryos with 2 pronuclei at the time of injection than in those with 1 (36.4 vs 17.9%). After 7 d in culture, 41.5% of morulae derived from zygotes with 2 pronuclei and 29.97% of thsoe derived from zygotes with 1 pronucleus showed signs of transgene expression. At this stage, significantly more morulae expressed the pbActinLacZ than the pSVON transgene (43.8 vs 25.8%). More than 80% of putative transgenic morulae or blastocysts showed evidence of mosaicism. These results demonstrate that IVM/IVF porcine embryos are able to develop in culture and express a microinjected transgene.

Journal Article↗
PubMed2001

Oxygen consumption of single bovine embryos probed by scanning electrochemical microscopy.

Oxygen consumption of individual bovine embryos was noninvasively quantified by scanning electrochemical microscopy (SECM). A probe microelectrode was used to scan near a single embryo surface in a culture medium to monitor the oxygen reduction current at 37 degrees C, under a water-saturated atmosphere of 5% CO2 and 95% air. The oxygen concentration profiles near the embryos were in good agreement with the theoretical spherical diffusion. When an embryo reached the stage of a morula with a 74-microm radius on day 6 after in vitro fertilization, the oxygen concentration difference (deltaC) between the bulk solution and the morula surface was 6.90 +/- 1.35 microM. The oxygen consumption rate (F) of the single morula was estimated to be (1.40 +/- 0.27) x 10(-14) mol s(-1). After the SECM measurement, the embryo was continuously cultured for another 2 days and grew to the stage of a blastocyst with a 100-microm radius. For the blastocyst, the deltaC values for the inner cell mass side and the trophoblast side were 16.40 +/- 1.83 and 9.14 +/- 1.68 microM, respectively. The oxygen consumption rate of the blastocyst was found to be in the range of (2.50 +/- 0.46) x 10(-14) mol s(-1) < F < (4.49 +/- 0.50) x 10(-14) mol s(-1). We have carried out SECM measurements for 19 embryos, and the results were compared in detail with these from an optical microscopic observation. The deltaC values for the morulae on day 6 after in vitro fertilization were strongly related to the morphological embryo quality. The morulae showing a larger deltaC value developed into blastocysts of a larger size, and the deltaC value after the subsequent 2 days of cultivation was found to be increased.

Animals↗