Glucose utilization by components of the mouse conceptus during early embryogenesis.

Mouse conceptuses were collected from female mice between day 6.5 and day 9.5 of pregnancy, dissected into their component parts and incubated for 2.5 h at 37 degrees C in droplets of Hepes-buffered medium containing 1 or 5 mmol glucose l-1 supplemented with 0.33 mmol pyruvate l-1 plus either 1 or 5 mmol (DL) lactate l-1 under oil. Glucose disappearance and lactate appearance were measured enzymatically at the end of incubation. High glucose concentration doubled utilization of this substrate per microgram of embryonic protein, but the change in lactate concentration had no effect on glucose turnover. Over the whole period of development studied, tissue from the ectoplacental cone exhibited the lowest rate of glucose turnover of all tissues isolated. At day 8.5, there was little difference between yolk sac and embryonic tissues, but by day 9.5, the yolk sac had a higher rate of utilization of glucose than did embryonic tissues. By this time, the embryonic tissues had started to show some metabolic differentiation, with head and visceral tissue exhibiting 20% higher turnover of glucose than that of body tissue. Overall, the rate of glucose utilization fell as development progressed and the estimate of the relative rate of glucose utilization on day 9.5 was half the value on day 6.5.

From uncommitted to committed embryonic cells: opportunities for their study and manipulation.

This paper offers a broad review of the methodologies used for the study of embryonic development and for the manipulation of embryonic cells. It was written in the context of the silver anniversary of the Australian Society for Reproductive Biology and places particular emphasis on the achievements of Australian scientists in this field. It has attempted to place recent advancements in some historical perspective, to examine the present "state of the art' in reproductive technologies and to consider future directions.

The metabolism of glutamine by the preimplantation sheep conceptus and its interaction with glucose.

The metabolism of glutamine and glucose, separately and in combination, by the sheep conceptus recovered on Days 2, 6, 13, 15, 17, and 19 of pregnancy was assessed over 2.5 h. At Day 2, the production of CO2 from glutamine was similar to that from glucose, with additive effects seen when both substrates were present. Between Day 2 and Day 6, there was a three-fold increase in glucose oxidation but no change in the oxidation of glutamine. From Day 13 to Day 19, the oxidation of glutamine was relatively high in embryonic tissue, low in trophoblastic tissue and intermediate in the yolk sac but in all tissues decreased as development progressed. Over this latter period the oxidation of glutamine was reduced to approximately 50% by the addition of glucose to the medium but glucose oxidation was unaffected by the addition of glutamine. At the early stages of development, the incorporation of substrate carbon from glutamine was less than that from glucose but in each case, incorporation into the acid-insoluble macromolecular fraction increased 2-3 times between Day 2 and Day 6. Incorporation of glutamine into the Day-17 and Day-19 conceptus was also measured; embryonic tissue exhibited the highest rate of incorporation and trophoblastic tissue the lowest. Incorporation was lower on Day 19 than on Day 17 and the proportion of carbon isolated in the acid-insoluble fraction represented 20% of the total incorporated. At no time did the addition of glucose to the medium alter incorporation of glutamine into either embryonic tissue or extraembryonic membranes.

Catabolic utilization of glucose by the sheep conceptus between days 13 and 19 of pregnancy.

The production of carbon dioxide and lactate from glucose by sheep embryos and samples of extraembryonic membranes was measured during a 2.5 h incubation period. Both embryos and their membranes were active in the glycolytic and oxidative utilization of glucose and, in general, the utilization of glucose per unit weight fell as development progressed from Day 13 to Day 19 of pregnancy. Both oxidation of glucose and glycolysis by the extraembryonic tissues, expressed as activity per microgram dried tissue, fell progressively with development. The rate of decline in CO2 production was greater than the rate for glycolysis and, as a consequence, the contribution of glycolysis to the estimated energy yield from the catabolism of glucose rose with time. In the embryo, both glucose oxidation and glycolysis peaked on Day 15 with estimates of adenosine triphosphate (ATP) production from glucose per microgram dried tissue on this day being 50% above those on Day 13 and 100% above those on Day 17. In general, the estimated yields of ATP from glucose were similar for structures of the same developmental age except that, at Day 19, it was calculated that the rate of ATP production by embryos was double that by the extraembryonic membranes. In incubations using 5.56 mM glucose as sole exogenous energy source, glucose turnover by embryos and embryonic membranes tended to be higher in a bicarbonate-buffered medium than in HEPES (4-(2-hydroxyethyl)-1-piperazincethane sulfonic acid) and phosphate-buffered media. As a result, the estimate of ATP yield plus the contribution of oxidative pathways to this yield were significantly higher in this medium than in the others. Glucose turnover by the embryo and its membranes in bicarbonate-buffered medium containing 0.56 mM glucose plus the alternate substrates, lactate and pyruvate, was severely depressed. Further experiments using samples of trophoblast and yolk sac indicated that both reduction in glucose concentration and the presence of the other substrates contributed to this suppression. Furthermore, an interaction between these factors was evident with the effects of alternative substrates being exaggerated when glucose concentration was low.

Oxidative utilization of glucose, acetate and lactate by early preimplantation sheep, mouse and cattle embryos.

The production of radiolabelled CO2 from [U-14C]glucose, [1-14C]lactate, and [U-14C]acetate was used to study the oxidative metabolism of embryos recovered from sheep, mice and cattle. Sheep embryos showed an increasing capacity to oxidize glucose after the 4- to 8-cell stage and oxidative turnover of this substrate at the blastocyst stage was four times that at the early stages. Decarboxylation of carbon-1 of lactate followed a pattern similar to that seen for glucose oxidation, but acetate oxidation was low and did not follow the trends with development seen for the other substrates. Furthermore, estimates of incorporation of acetate into the macromolecules of sheep embryos were low compared with similar estimates for glucose and, unlike glucose, did not increase with development. Oxidation of all three substrates by mouse embryos increased with development but the rate of CO2 production from acetate was low compared with that from the other substrates. A combination of lactate or glucose with acetate had no influence on the utilization of acetate by mouse morulae/early blastocysts, nor did acetate influence utilization of the other substrates. Cattle morulae/early blastocysts also produced more CO2 from glucose and lactate than from acetate and the incorporation of carbon from acetate into the macromolecules of these embryos was less than from glucose. Overall, cattle embryos showed the greatest ability to metabolize acetate but were not as effective in using the other substrates as either sheep or mouse embryos at the same stage.

Effect of oxygen concentration on the incorporation of glucose by the sheep conceptus between days 13 and 19 of pregnancy.

Embryos and extraembryonic membranes recovered from the sheep conceptus on Days 13, 15, 17 and 19 of pregnancy were incubated in medium containing glucose as sole energy substrate. In all components of the conceptus 60-70% of substrate carbon incorporated was recovered in the non-glycogen acid-soluble fraction, 25-30% in non-glycogen macromolecules and 4-8% in the glycogen pools. At all stages of development examined, embryonic tissue accumulated more glucose carbon into all fractions than did yolk sac which in turn was more active than trophoblast. After its appearance, the allantois was at least as active in glucose incorporation as embryonic tissue. Over the period of development examined, incorporation into all tissues of the conceptus fell progressively as pregnancy advanced and, by Day 19, total incorporation was about 60% of the initial value for each component. Reduction in oxygen concentration generally depressed incorporation into all intracellular carbon pools. The most consistent and significant effects were recorded for the two non-glycogen pools where incorporation fell, on average, by 30-40% when O2 concentration was reduced to 1%. Most of the response observed was due to a drop in O2 concentration from 20 to 5% with smaller additional effects when the O2 was further reduced to 1%. Incorporation into all pools isolated tended to follow a similar pattern and incorporation into the three macromolecular components, expressed as a percentage of total incorporation, remained unchanged as O2 concentration was reduced.

Contribution of the pentose phosphate pathway to glucose utilization by preimplantation sheep embryos.

The activity of the pentose phosphate pathway of glucose metabolism in early sheep embryos and in the structures of the advanced conceptus from Day 13 to Day 19 of pregnancy was measured quantitatively during a 2.5-h incubation with glucose as sole energy source. For embryos during cleavage, activity of this pathway accounted for 6-9% of total glucose utilized. The proportion of glucose metabolized through the pentose pathway fell progressively with development and by Day 19 represented 1-2% of glucose turnover. However, total turnover of glucose increased eight fold between the 2-cell and blastocyst stage and the amount of glucose processed through the pentose pathway increased over this time despite the fall in the proportion utilized in this way. In contrast, glucose turnover by the advanced embryo and its extra embryonic membranes progressively decreased as the structures developed. As a result, estimates of the amount of glucose utilized through the pathway per microgram dried weight per hour declined to low values at Day 19 following the peak in activity at about the time of blastulation. Trophoblast and yolk sac processed less glucose through the pentose pathway per microgram dried weight than embryonic tissue but the allantois was similar to the embryo. Overall, the pentose pathway accounted for a relatively constant proportion of the CO2 produced from glucose under these experimental conditions with values generally between 15 and 20% of total CO2 produced. When activities in the components of the advanced conceptus were expressed as the total amount of glucose processed through the pathway per hour, turnover in the embryo, allantois and yolk sac increased progressively with time. By contrast, there was a substantial trough in the activity of the trophoblast on Day 17 of pregnancy.

Oxidation of [U-14C]acetate by the sheep conceptus between days 13 and 19 of pregnancy.

Acetate metabolism by the sheep conceptus was assessed by measuring CO2 production during a 2.5-h incubation of embryos and samples of the extraembryonic membranes in HEPES-buffered media containing 1.12 mM [U-14C]acetate. The rate of oxidation of acetate by embryonic tissue showed little change between Days 13 and 15 of pregnancy but greatly decreased by Days 17 and 19. By contrast, oxidation of the substrate by the trophoblast increased substantially with development and was five times the early rate by Day 19. Oxidation of acetate by the yolk sac also increased 4-fold between Days 17 and 19. The addition of glucose to incubations of extraembryonic membranes resulted in some reduction in the oxidation of acetate by the yolk sac and allantois but had little effect on the trophoblast. At Days 13 and 15, the rate of oxidation of acetate by the embryonic disc was 6-7 times that by the trophoblast. As development progressed, this situation was reversed and by Day 19 the trophoblast metabolized more than five times the amount of acetate per microgram than did the Day-19 embryo. Although acetate metabolism by yolk sac and allantois on Day 17 was low, its metabolism by the yolk sac increased to values similar to those for the trophoblast at Day 19 but its utilization by the allantoic membrane remained low. Comparison of the estimates of ATP generated from acetate by these tissue with those published for glucose demonstrates that acetate is much less effective than glucose for the provision of metabolic energy.

Incorporation of substrate carbon from [U-14C]acetate by the sheep conceptus recovered from the uterus on days 13 to 19 of pregnancy.

Of the substrate carbon incorporated from 1.12 mM [U-14C]acetate as sole energy substrate during a 2.5-h incubation, a large proportion was isolated in the acid-soluble fraction. Although there was no significant change over time in the rate of entry into this pool for embryos, the rate of accumulation by the trophoblast and yolk sac increased as development progressed. At Days 13 and 15 of pregnancy, incorporation of acetate into the acid-insoluble fraction of embryos accounted for almost half the total label accumulated. The rate of this incorporation fell rapidly over time and by Day 19 less than 30% of carbon accumulated was in this fraction. By contrast, the rate of incorporation into this fraction by trophoblastic tissue was low at Day 13 but rose dramatically as development progressed. Incorporation by the yolk sac into acid-insoluble components also rose with time. At the early stages of pregnancy studied, lipid synthesis accounted for the majority of acetate carbon accumulated by the conceptus in the acid-insoluble pool. At later stages of development, incorporation into lipids constituted a minor pool of acetate carbon. Some acetate carbon was found in the glycogen fraction of the conceptus. The rate of incorporation into the acid-soluble glycogen fraction by embryos was constant throughout the period studied. By contrast, trophoblast increased its rate of incorporation markedly into both acid-soluble and acid-insoluble glycogen pools as did the yolk sac from Day 17.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of culture from the zygote stage on the metabolism of glucose and glutamine by 2-cell embryos and blastocysts recovered from outbred or F1 hybrid female mice.

The oxidation and incorporation of glucose and glutamine by embryos derived from cultured zygotes was compared with the utilization of these substrates by embryos recovered directly from the reproductive tract of pregnant females. The oxidation of glutamine was greater at the blastocyst stage than at the 2-cell stage. Embryos derived from outbred females (Qs) were less active in the oxidation of glutamine than those from hybrid (B10D2F1) females and development in culture was detrimental to this oxidation, especially in blastocysts from the outbred stock. The oxidation of glutamine was stimulated by the presence of glucose at the 2-cell stage but reduced by its presence at the blastocyst stage. Maternal genotype had no effect on the oxidation of glucose at either the 2-cell or blastocyst stage, and only at the blastocyst stage was there evidence of a detrimental effect of culture. The oxidation of glucose was stimulated by the presence of glutamine at the 2-cell stage but depressed by its addition at the blastocyst stage. Incorporation of glutamine increased with development, but this was reduced at the blastocyst stage by development in culture, especially if the blastocysts were derived from outbred females. Incorporation of glucose also increased with development. At the 2-cell stage, culture reduced incorporation of this substrate, especially into the acid-soluble fraction of embryos from outbred females. In blastocysts, incorporation of glucose into the acid-insoluble fraction was depressed by culture and in embryos from outbred females. In contrast to glucose oxidation, incorporation of glucose into the acid-soluble fraction was reduced by the addition of glutamine at the 2-cell stage but increased by its addition at the blastocyst stage.

Glycolysis and glucose oxidation by the sheep conceptus at different oxygen concentrations.

The effect of changes in oxygen concentration on the catabolic utilization of glucose by the sheep conceptus at selected periods between Days 3 and 19 of preimplantation development was examined by estimating the production of CO2 and lactate from [U-14C]glucose during a 2.5-h culture in vitro in the presence of 20%, 5% and 1% O2. In general, lowering O2 significantly altered the catabolism of glucose with a changing pattern of response depending on the stage at which the conceptus was explanted. For embryos at Days 3 and 6 post insemination, reduced O2 caused no significant change in oxidative utilization of glucose and a small decrease in conversion of the substrate to lactate. By contrast, lowering O2 concentration during incubation of the structures of the advanced conceptus from Day 13 through to Day 19 of pregnancy significantly restrained oxidative utilization of glucose but stimulated its conversion to lactate. The effects of these changing levels of O2 on the generation of energy from glucose in the form of ATP was estimated. Except for the Day-13 conceptus, reduction in O2 concentration had little influence on the calculated amount of ATP produced from glucose, with glycolysis making up the deficit in energy production when reduced O2 inhibits oxidation of the substrate at the later stages of development. At Day 13, the switch in the metabolism of glucose to glycolysis is not fully effective and energy production fell as O2 concentration was reduced. The results indicate a major shift towards dependence by the preimplantation sheep conceptus on the glycolytic pathway for energy generation from glucose as development progresses. This move to glycolysis is increased by low O2 concentration. As a low concentration of O2 most probably exists in the lumen of the sheep uterus, the results indicate that, in utero, the energy required for the rapid growth of the conceptus depends progressively more on glycolysis than oxidative metabolism of glucose. The finding that the Day-13 conceptus has not fully adapted to this method of ATP generation at low O2 concentrations may make it especially vulnerable during development in utero.

Metabolism of glucose by vesicles derived from sheep trophoblast and embryonic discs.

Samples of trophoblast recovered from the sheep conceptus on Day 13 of pregnancy formed spherical vesicles during culture in medium 199. These continued to expand and increase in dry weight over the next 6 days in vitro. After 6 days' culture, the metabolism of glucose by these vesicles was compared with that of Day-13 and Day-19 fresh trophoblastic tissue. The production of CO2 and lactate by vesicles was similar, although not identical, to production by Day-13 fresh tissue and did not exhibit the marked decrease in glucose catabolism seen in Day-19 trophoblast. The tissue from vesicles reacted to reduction in oxygen tension in a manner similar to the reaction of fresh tissue, with decreased glucose oxidation and increased glycolysis. The activity of the pentose phosphate pathway in vesicles was higher, and the activity in Day-19 fresh tissue was much lower, than that in Day-13 fresh trophoblast. Incorporation of glucose into the intracellular biochemical pools by vesicles was similar to incorporation into Day-13 fresh tissue. Limited observations were also made with vesicles derived from embryonic disc. Production of CO2 by these vesicles was intermediate between that detected in fresh Day-13 and Day-19 embryonic tissue. There were not significant differences in lactate production between fresh and cultured samples of embryonic tissue. These results show that vesicles formed in vitro remain metabolically active but do not mimic the biochemical changes seen in the tissue during development in vivo.

Some effects of genotype and composition of the culture medium on the development of mouse zygotes in vitro.

The effects of EDTA and the presence of glucose and glutamine in CZB medium on the development of mouse zygotes of different genotype were investigated. Although 30-80% of zygotes (depending on the cross) passed the 2-cell stage in EDTA-free medium, the addition of a low concentration of EDTA was necessary in these experiments to obtain blastocysts in culture. In reciprocal crosses between outbred (Qs), inbred (DBA/2) and hybrid (B10D2F1) stock, there was evidence of a strong influence of the maternal genome on zygote development, with those from B10D2F1 females performing best irrespective of sire. A paternal influence on development was also evident but the most successful sire varied with the genotype of female used and reciprocal crosses differed greatly in the ability of the resultant zygote to develop in culture. For zygotes recovered from Qs females, CZB medium containing glucose and glutamine supported development to the blastocyst stage better than did medium devoid of these substrates. Tests with embryos from B10D2F1 females indicated that the presence of glucose for the whole or for part of the incubation period stimulated blastocyst development. However, the addition of glutamine to the medium in these tests had no significant effect on the development of blastocysts.

Assisted fertilization of mouse oocytes and preliminary results for human oocytes using zona drilling.

The zona-drilling procedure was investigated in mouse oocytes prior to a study on human oocytes. The procedure involved the injection of 5-nl volumes of acidic Hepes-buffered medium at pH 2.5 using a microinjection instrument. Zona-drilled mouse oocytes had significantly higher rates of fertilization (60/99; 61%) than zona-intact oocytes (6/103; 6%) at an insemination concentration of 1 x 10(4) sperm/ml (P less than 0.001). The procedure did not induce parthenogenetic activation of oocytes and more than 97% of zygotes developed to the blastocyst stage. A similar rate of live progeny was observed when zona-drilled (38.0%) and control embryos (38.5%) were transferred to pseudopregnant recipients. Chromosome analyses were performed on zona-intact, zona-free, and zona-drilled oocytes inseminated with varying concentrations of sperm and analysed at the first cleavage division. Zona-free oocytes had high rates of polyploidy (greater than or equal to 40%) with varying insemination numbers but the zona-drilled oocytes did not reveal significant increases in the rate of polyploidy or aneuploidy when compared to controls. In the human studies, zona-drilled oocytes achieved higher rates of fertilization than zona-intact oocytes, with sperm numbers as low as 1 x 10(4)/ml (6/8; 75%). Polyspermic fertilization was observed in 1/2 and 2/6 of fertilized oocytes inseminated with 1 x 10(5) and 1 x 10(4) sperm/ml, respectively. With the low sperm concentration 2/4 of those which were normally fertilized developed to healthy blastocysts. These studies suggest that the zona-drilling technique as described can be performed without apparent harm to oocytes and generate normal embryos.(ABSTRACT TRUNCATED AT 250 WORDS)

Oxidative metabolism of energy substrates by preimplantation mouse embryos in the presence of platelet-activating factor.

The effects of exogenous platelet-activating factor (PAF, 0.0186-18.6 microM) on the production of CO2 from [U-14C]glucose and [1-14C]lactate by mouse embryos in vitro were investigated. Two-cell embryos displayed significant dose-dependent responses for both energy substrates. The maximal response was observed at 9.3 microM-PAF for glucose metabolism and 1.86 microM-PAF for lactate with increases of 62% and 18%, respectively, over control treatments. After culture from the 2-cell stage for 72 h in the presence of PAF, the resulting blastocysts exhibited a significant dose-dependent increase in metabolism of lactate. It was also apparent that such embryos were not desensitized to PAF as demonstrated by a further enhancement of the metabolic response after re-exposure to PAF. The specificity of action of PAF was confirmed by the absence of any effect on the oxidative metabolism of glucose by lyso-PAF (a catabolite of PAF) over a concentration range of 0.0202-20.2 microM and by the demonstration that SRI 63-441 (a PAF-receptor antagonist) significantly reduced the amount of CO2 produced from glucose in response to 9.3 microM-PAF and abolished the effect on lactate metabolism in response to 1.86 microM-PAF. These results demonstrate a specific, direct influence of exogenous PAF on the oxidative metabolism of glucose and lactate by the preimplantation mouse embryo and suggest an autocrine role for embryo-derived PAF in early pregnancy.

Implantation potential and fetal viability of mouse embryos cultured in media supplemented with platelet-activating factor.

Supplementation of culture media with PAF failed to increase the proportion of 2-cell mouse embryos which developed through to the expanded blastocyst stage after 72 h of culture in vitro. Culture of embryos with 1.86 microM-PAF, however, significantly increased the number of cells in expanded blastocysts. Culture in vitro with 0.186 or 1.86 microM-PAF significantly increased the ability of embryos to implant upon transfer to pseudopregnant recipients. The specificity of this effect was demonstrated by its inhibition by the specific PAF-receptor antagonist, SRI 63-441. Of the embryos which implanted, there was no difference in the proportions of PAF-treated or control embryos which resorbed nor was there any effect of PAF on fetal weight or crown-rump length measurements. Embryos cultured with 1.86 microM-PAF did have slightly heavier placentae than their respective controls. These results demonstrate the ability of exogenous PAF to enhance the viability of preimplantation mouse embryos cultured in vitro and support the hypothesis that a major site of the action of PAF during early pregnancy is the embryo itself.

Participation of glucose in the synthesis of glycoproteins in preimplantation mouse embryos.

Electrophoretic separation of solubilized embryos incubated for 24 h in the presence of [U-14C]glucose indicated incorporation of glucose carbon into a number of protein bands. Treatment of nitrocellulose blots of electrophoretograms with glucosidases had no effect on incorporated counts, confirming that the labelled bands were not due to protein bound glycogen. Furthermore, addition of 0.1 microgram mL-1 tunicamycin to the incubation medium virtually eliminated incorporation of glucose into the protein bands but had no effect on the pattern or rate of incorporation of labelled amino acids in parallel experiments. Also the pattern of labelling of protein by glucose was reflected in the pattern of binding of Con A to the nitrocellulose blots. There were quantitative and qualitative changes in labelling as development progressed. For embryos cultured from the 2-cell stage, a small amount of label was incorporated in two major bands at relative mobility (Mr) 69 and 97 K. With culture from the 8-cell stage, three additional major bands (33, 44 and 56 K) were labelled. Embryos cultured from the morula stage showed a different profile of incorporation; there was much more active labelling, and eight major and a number of minor radioactive bands were identified. Whilst tunicamycin suppressed glucose incorporation into glycoproteins and inhibited compaction of embryos, it had little effect on other parameters of metabolism during incubation in its presence for 24 h. No significant effects of the metabolite on protein synthesis, glycogen storage, lactate production or overall macromolecular synthesis were evident. By contrast, the anabolic metabolism of embryos decompacted by long periods of exposure to tunicamycin was severely reduced although glycolysis was still unaffected. Amphomycin at very high concentration (500 micrograms mL-1) was toxic to embryos but at concentrations up to 250 micrograms mL-1 had no effect on compaction and development of blastocysts. Addition of monensin to the incubation medium [16 micrograms mL-1] did not interfere with the development of either 2-cell or 8-cell embryos to blastocysts.

Ultrastructural observations on gamete interactions using micromanipulated mouse oocytes.

Cumulus-free mouse oocytes were subjected to zona opening by cracking with microhooks (ZC) or acid drilling (ZD) and fixed 30-90 min after insemination (10(5) pre-capacitated motile sperms/ml). Ultrastructural observations were made on serially thin-sectioned oocytes: 15 ZC and 12 ZD. The zona lesion in ZC oocytes was a clean cut, whereas in ZD oocytes it formed a patchy area of partial zona loss, with reduced microvillar height on the underlying oocyte surface. Spermatozoa were observed within the perivitelline space and partially fusing with the oocyte after 30 min in both situations. Only acrosome-reacted sperm heads were observed to fuse: acrosome intact forms were generally in contact with the zona pellucida, either with the inner or outer surface. Acrosome-intact spermatozoa were also observed deeply embedded in the zona matrix, possibly indicating surface enzyme activity preceding the membrane fusion events of the acrosome reaction proper. The observations are consistent with the need for spermatozoa to make contact preferentially with the zona pellucida during the course of the acrosome reaction.

Development of sheep preimplantation embryos in media supplemented with glucose and acetate.

Two experiments were conducted to examine the effect of supplemental glucose (G; 1.5 mM) and/or acetate (A; 0.5 mM) on the development of early sheep embryos to blastocysts when cultured in vitro in glucose-free synthetic oviductal fluid (SOF)+sheep serum or bovine serum albumin (BSA). In Experiment 1, 2- to 4-cell, 8- to 16-cell and >16-cell embryos were cultured in SOF, SOF+G, SOF+A or SOF+G+A. All media were supplemented with 10% sheep serum. In addition, embryos were cultured in either microdrops under polysiloxane oil or in multiwell dishes. Overall, development to the blastocyst stage was 3%, 30% and 68% for 2- to 4-cell, 8- to 16-cell and >16-cell stages, respectively, suggesting that an 8-cell developmental block existed under our culture conditions. Glucose supplementation had little effect on embryo development, and no overall effect was observed from the addition of acetate. In Experiment 2, 8- to 16-cell embryos were cultured in SOF or SOF+G, both supplemented with BSA. Development to the blastocyst stage was 25% and 18%, respectively. The results show that the presence of glucose or acetate did little to enhance embryonic development in our incubation systems. Further work is required to evaluate fully the energy requirements for development of the early sheep embryo.