Leigh syndrome: clinical features and biochemical and DNA abnormalities

The morphology and number of cells in the trophectoderm (TE) and inner cell mass (ICM) of buffalo blastocysts derived from in vitro fertilization and cultured in the presence or absence of insulin-like growth factor-I (IGF-I) were analyzed by differential fluorochrome staining technique. The total cell number (TCN), TE number, and ICM cell number were significantly higher in blastocysts developed in vitro in the presence of IGF-I as compared to blastocysts developed without IGF-I (P < 0.01). It was observed that the buffalo blastocyst took 5-9 days postfertilization to develop in vitro. In order to correlate the time required for blastocyst development and the allocation of cells to TE and ICM, blastocysts were designated as fast (developing on or before day 7) or slow (developing after day 7). The TCN, TE, and ICM cells of fast-developing blastocysts cultured in the presence of IGF-I were significantly higher than slow-developing blastocysts (P < 0.01). The blastocysts developed on day 6 had a mean total cell number 118.6 +/- 21.4, which significantly decreased to 85.6 +/- 17.4, 62.0 +/- 14.5, and 17.0 +/- 4.0 on days 7, 8, and 9, respectively (P < 0.05). Normal development of buffalo embryo showed that, on average, embryos reached compact morula stage at the earliest between days 4.5-5.5. Blastocysts developed, at the earliest, between days 5.0-6.0, and it took them, on average, 6.5 days to hatch from the zona pellucida. TCN, TE, and ICM increased three times from morula to blastocyst; however, the proportion of ICM to TCN remained the same, in both embryonic stages. TE approximately doubled in hatched blastocysts, as compared to unhatched blastocysts (P < 0.05). However, ICM cells were decreased. The time required for development of parthenogenetic blastocysts was observed to be greater as compared to in vitro fertilized (IVF) blastocysts. The total cell number of parthenogenetic blastocysts was 100.8 +/- 11.3, including 59.2 +/- 8.4 cells of TE and 42.1 +/- 6.9 cells of ICM.     

The high affinity state of inositol 1,4,5-trisphosphate receptor is a functional state

Inositol 1,4,5-trisphosphate (InsP3) is a second messenger responsible for the rapid and discontinuous release of Ca2+ from intracellular stores. In this study, the effects of the sulfhydryl reagent thimerosal were investigated on Ca2+ mobilization and on InsP3 binding. Thimerosal was shown to release Ca2+, in a dose-dependent manner, with an EC50 of 135.8 +/- 5.2 microM, from bovine adrenal cortex microsomes. Thimerosal-induced Ca2+ release was not prevented by heparin (250 micrograms/ml), ruling out a participation of InsP3 receptor in that effect. The slow rate of thimerosal-induced Ca2+ release rather suggested an inhibition of microsomal Ca2+ ATPase. At submaximal concentration, thimerosal (100 microM) was also shown to potentiate the release of Ca2+ induced by InsP3. Dose-response experiments revealed that thimerosal enhanced the apparent affinity of InsP3 by a factor 2.21 +/- 0.28, without modifying the maximal amount of Ca2+ released by InsP3. Thimerosal also enhanced, in a dose-dependent manner, [3H]InsP3 binding to adrenal cortex microsomes (EC50 = 43.3 +/- 7.6 microM). A similar effect was also observed on [3H]InsP3 binding to solubilized receptors, suggesting a direct modification of the receptor protein by thimerosal. The effects of thimerosal on Ca2+ release and [3H]InsP3 binding were abolished in the presence of the reducing agent dithiothreitol (1 mM), suggesting a modification by thimerosal of specific thiol groups on these microsomal proteins. Scatchard analysis revealed that thimerosal (100 microM) increased InsP3 receptor affinity by 1.87 +/- 0.26-fold. Kinetic analysis indicated that this increased affinity was due to an enhancement of InsP3 association rate constant. The concomitant increases of binding affinity and Ca2+ releasing potency suggest that the high affinity state of InsP3 receptor is a functional state.     

Development of pig oocytes activated by stimulation of an exogenous G protein-coupled receptor

This study determined whether stimulation of a G protein-coupled receptor could initiate the events that occur at fertilization in pig oocytes and, if so, whether the activated oocytes were competent to form blastocysts. After maturation for 30 h, oocytes received microinjections of mRNA encoding the rat M1 muscarinic receptor, a G protein-coupled acetylcholine (ACh) receptor. Oocytes were then incubated for an additional 15 h to complete maturation of oocytes and translation of microinjected mRNA, and they were subsequently cultured in the presence of ACh. ACh treatment of these oocytes triggered pronuclear formation (50.4%) as well as cortical granule exocytosis. SDS-PAGE showed that mRNA-microinjected oocytes treated with ACh were activated (61.1%), as characterized by the appearance of the 22-kDa polypeptide derived from dephosphorylation of the 25-kDa precursor. Furthermore, after being cultured in a ligated pig oviduct for 6 days, 17.4% of treated oocytes developed to the compact morula or blastocyst stage. Transmission electron microscopy revealed that blastocysts recovered from ligated oviducts contained reticulated nucleoli with fibrillar cores surrounded by fibrillar and granular components. In addition, mitochondria in the blastocysts were dispersed throughout the cytoplasm and contained numerous transverse cristae. These results show that pig oocyte activation mediated by a G protein-coupled signal transduction system can signal a series of intracellular changes that lead to activation events associated with fertilization. Furthermore, oocytes activated through this pathway showed preimplantation development consistent with normal development.     

Changes in ribosomal ribonucleic acid content within in vitro-produced bovine embryos

The abundance of 28S, 18S, and 5S rRNA was measured by Northern blot techniques applied to RNA samples extracted from bovine oocytes and preattachment embryos produced by in vitro procedures. Total RNA content was estimated by comparing the intensity of hybridization signals of 28S and 18S rRNA probes to embryo RNA samples and to standard curves generated from bovine ovary or bovine oviduct cell RNA. RNA content declined from the oocyte to the morula stage (2.4 +/- 0.3 ng/oocyte, 1.7 +/- 0.5 ng/1-cell embryo, 2.2 +/- 0.9 ng/2- to 4-cell embryo, 0.8 +/- 0.2 ng/6- to 8-cell embryo, and 0.7 +/- 0.2 ng/morula). A marked increase in RNA content, based on levels of hybridization to 28S and 18S rRNA, was observed in blastocysts, in which values averaged 5.3 +/- 0.6 ng/embryo. On a relative basis, 5S rRNA abundance followed a pattern similar to that of 28S and 18S rRNA across the early development period to the blastocyst stage.     

Effect of sulfhydryl oxidoreduction on permeability of cardiac tetrodotoxin-insensitive sodium channel

Effects of sulfhydryl oxidizing and reducing agents on permeability of the tetrodotoxin (TTX)-insensitive Na-channel were investigated in guinea-pig ventricular myocytes using the whole-cell patch-clamp technique. Mercury chloride (HgCl2) at 1-100 microM irreversibly blocked Na+ currents with no significant changes in the gating kinetics. In contrast, the hydrophilic sulfhydryl oxidizing agent, thimerosal at 50-100 microM little affected Na+ permeation through the Na-channel. The Hg2+-induced block of Na+ current could be readily reversed by 1,4-dithiothreitol (DTT), an agent that reduces disulfide bonds. These results indicate that the formation of sulfur-Hg-sulfur bridge is essential for Hg2+ block. Pretreatment with DTT prevented the Hg2+ block of Na+ current, whereas Zn2+ and Cd2+ retained their abilities to block Na+ current after DTT treatment. An application of Zn2+ or Cd2+ resulted in the restoration of Hg2+ sensitivity of the DTT-treated channel. A conformational model for the Na-channel with multiple free sulfhydryl groups and native disulfide bonds could account for our experimental data regarding the effects of sulfhydryl modifying agents on the channel permeability. We conclude that the cardiac TTX-insensitive Na-channel contains functionally important free sulfhydryl groups and disulfide bonds which are accessible from the extracellular side by an aqueous pathway. These sulfhydryls would be capable of modulating the Na-channel permeability by affecting the conformation of channel pore region.     

Development of calcium signalling mechanisms during maturation of human oocytes

Sperm-induced Ca2+ signals mediate the events of oocyte activation at fertilization. In this study, the development of mechanisms involved in the generation of Ca2+ signals in human oocytes was investigated. The thiol reagent, thimerosal, which induces oscillations of intracellular Ca2+ ([Ca2+]i) similar to those seen during fertilization, was used to mobilize Ca2+ in in-vivo matured, immature and in-vitro matured human oocytes. There was an increase in the sensitivity to thimerosal during maturation of human oocytes, with oocytes from small antral follicles being relatively insensitive, compared with those from luteinized follicles, which displayed a large spike followed by sustained oscillations in [Ca2+]i. These oscillations were inhibited by caffeine which suggests that they were mediated by the inositol trisphosphate receptor Ca2+ release system. When immature oocytes were cultured in vitro they acquired the capacity to undergo a single large spike in [Ca2+]i, however, subsequent sustained oscillations were not observed, indicating that these oocytes failed to develop fully competent Ca2+ signalling mechanisms during culture in vitro. This finding may be a key factor in the poor developmental competence of in-vitro matured human oocytes.     

Effect of cytochalasin B and cycloheximide on the activation rate, chromosome constituent and in vitro development of porcine oocytes following parthenogenetic stimulation

Activation rate, chromosome constituent and developmental pattern of porcine oocytes was examined in the presence and absence of cytochalasin B and cycloheximide following parthenogenetic stimulation. Treatment with cycloheximide after ethanol or Ca2+ ionophore treatment increased the incidence of activation. The percentage of oocytes with two or more female pronuclei was higher (P < 0.05) in oocytes treated with cytochalasin B than in control or cycloheximide-treated oocytes. Treatment with both electrical stimulation and cytochalasin B increased the incidence of diploid chromosome spreads, and accelerated development to the morula and blastocyst stage compared with the control and cycloheximide-treated groups, suggesting a role of ploidy in the development of parthenote.     

Effect of thimerosal on cytosolic calcium and phosphatidylserine synthesis in Jurkat T cells

1. We investigated the effect of the thiol reagent, thimerosal on calcium movements in the Jurkat T cell line. 2. Thimerosal induced a rise in cytosolic Ca2+ concentration due both to a release of Ca2+ from intracellular stores and a Ca2+ influx. 3. Thimerosal, released Ca2+ from the same intracellular stores than CD3 mAb and ionomycin. 4. Emptying the Ca2+ intracellular stores was accompanied by a marked decrease of phosphatidylserine synthesis indicating that phosphatidylserine synthesis occurs within or close to the endoplasmic reticulum Ca(2+)-stores as previously described in CD3-, ionomycin- or Ca(2+)-ATPase inhibitor-treated lymphocytes.     

Glucose transporter GLUT3: ontogeny, targeting, and role in the mouse blastocyst

The first differentiative event in mammalian development is segregation of the inner cell mass and trophectoderm (TE) lineages. The epithelial TE cells pump fluid into the spherical blastocyst to form the blastocyst cavity. This activity is fuelled by glucose supplied through facilitative glucose transporters. However, the reported kinetic characteristics of blastocyst glucose transport are inconsistent with those of the previously identified transporters and suggested the presence of a high-affinity glucose carrier. We identified and localized the primary transporter in TE cells. It is glucose transporter GLUT3, previously described in the mouse as neuron-specific. In the differentiating embryo, GLUT3 is targeted to the apical membranes of the polarized cells of the compacted morula and then to the apical membranes of TE cells where it has access to maternal glucose. In contrast, GLUT1 was restricted to basolateral membranes of the outer TE cells in both compacted morulae and blastocysts. Using antisense oligodeoxynucleotides to specifically block protein expression, we confirmed that GLUT3 and not GLUT1 is the functional transporter for maternal glucose on the apical TE. More importantly, however, GLUT3 expression is required for blastocyst formation and hence this primary differentiation in mammalian development. This requirement is independent of its function as a glucose transporter because blastocysts will form in the absence of glucose. Thus the vectorial expression of GLUT3 into the apical membrane domains of the outer cells of the morula, which in turn form the TE cells of the blastocyst, is required for blastocyst formation.     

Effects of glycosaminoglycans on the development of in vitro-matured and -fertilized porcine oocytes to the blastocyst stage in vitro

We examined the effects of four glycosaminoglycans (GAGs) on the development of in vitro-matured (IVM) and -fertilized (IVF) porcine oocytes to the blastocyst stage. IVM and IVF oocytes were cultured in Whitten's medium supplemented with hyaluronic acid, chondroitin sulfate A, dermatan sulfate, or heparin at 38.5 degrees C in an atmosphere of 5% CO2 in humidified air for up to 6 days. After 2 days in culture, 28-34% of the inseminated oocytes cleaved to the 2- to 8-cell stage, and the GAGs showed no significant effect on development. After 6 days in culture, blastocysts were observed in all groups. The percentage of blastocysts was significantly higher in hyaluronic acid-supplemented medium (14%) than in dermatan sulfate-supplemented (5%), heparin-supplemented (2%), or nonsupplemented (2%) media. In addition, the percentage of blastocysts was significantly higher in chondroitin sulfate A-supplemented medium (11%) than in heparin-supplemented and nonsupplemented media, although the number of blastocysts in chondroitin sulfate A was not significantly different from that in hyaluronic acid- and dermatan sulfate-supplemented media. There were no significant differences in the mean number of nuclei per blastocyst cultured in any group. The effects of hyaluronic acid and chondroitin sulfate A on development to the blastocyst stage was examined at various concentrations. After 6 days in culture, development of IVM and IVF oocytes to the blastocyst stage was best supported in 0.5 mg/ml hyaluronic acid-supplemented (17%) and in 0.1 or 0.5 mg/ml chondroitin sulfate A-supplemented (10% or 9%, respectively) media. It is concluded from these results that hyaluronic acid and chondroitin sulfate A supported the development of porcine oocytes matured and fertilized in vitro to the blastocyst stage.     

Increased site-specific phosphorylation of tyrosine hydroxylase accompanies stimulation of enzymatic activity induced by cessation of dopamine neuronal activity

The growth of ferret preimplantation blastocysts in vivo, collected between 156 and 240 hr post coitum, was investigated. A technique, combining immunosurgery and differential fluorochrome staining, was used to discriminate between inner cell mass (ICM) and trophectoderm (TE) cells. Using the stains propidium iodide and bisbenzimide (Hoechst 33342), the ICM was stained blue and the TE was stained pink. The ICM and TE counts for 90 blastocysts, respectively, averaged 25 and 63 at 156 hr and increased exponentially to 2077 and 4137 at 240 hr. The Box-Cox procedure was used for choosing a transformation that minimized the error sum of squares for a linear regression of Y (cell count) on X (time in hr). Logarithmic transformations of the ICM, TE and total cell count gave a good fit, but the following equations obtained by the Box-Cox procedure provided the best fit, where Y is cell count and X is time in hours. For inner cell mass: Y = [(176.06 + 2.45X)/-899.44 + 1]-3.33; trophectoderm: Y = [(301.38 + 14.48X)/-6863.42 + 1]-10; and total: Y = [(2266.97 + 17.0X)/-7837.21 + 1]-5. The R2 values were 0.73, 0.84, and 0.84, respectively. The exponential growth of the ferret embryo during the time interval that measurements were made fits the general pattern described for other mammalian embryos. This report is the first to characterize the pattern of cell allocation and growth in preimplantation blastocysts of the ferret, and the first such report for a carnivore. The pattern of in vivo development provides a standard for judging the quality of in vitro produced and matured ferret embryos and, concomitantly, a means to evaluate culture systems.     

U73122 blocked the cGMP-induced calcium release in sea urchin eggs

U73122, a phospholipase C inhibitor, dose dependently blocks the cGMP-induced Ca2+ release in sea urchin eggs and homogenates. U73122 inhibition was prevented by cotreatment with dithiothreitol (DTT), but DTT is ineffective when eggs or homogenates were pretreated with U73122. U73122 action is different from the other sulfhydryl reagents, thimerosal and N-ethylmaleimide, which cause Ca2+ release in egg homogenates at high concentration, but at lower concentration have no significant effect on cGMP-induced Ca2+ release. Histone, a reported NAD glycohydrolase (NADase) activator, was found to induce Ca2+ release in egg homogenates via the same pathway as the cGMP response, since histone-induced Ca2+ release is blocked by Rp-8-pCPT-cGMPS, a cGMP-dependent protein kinase (PKG) inhibitor, and nicotinamide, a NADase inhibitor. Histone-induced Ca2+ release is similarly blocked by U73122. The aminosteroid U73122 does not inhibit cADPR-induced Ca2+ release, which is significantly reduced by PKG inhibitors. Furthermore, U73122 has no significant effect on phorbol 12-myristate 13-acetate induced-cytoplasmic alkalinization in intact eggs, which depends on protein kinase C activity. These results suggest that U73122 does not act as a general serine-threonine protein kinase inhibitor, and the aminosteroid inhibition of the cGMP-induced Ca2+ release may interfere with ADP ribosyl cyclase activity.     

Thimerosal triggers meiosis reinitiation in oocytes of the Japanese clam Ruditapes philippinarum by eliciting an intracellular Ca2+ surge

Ovarian oocytes of the bivalve mollusc Ruditapes philippinarum are arrested during first meiotic prophase. Release from this blockade is triggered by the neurohormone serotonin (5HT or 5-hydroxytryptamine), which promotes germinal vesicle breakdown and drives these oocytes to a second arrest in metaphase I. 5HT action involves binding to a specific G protein-coupled receptor which results in a transient rise in IP3 and in the intracellular free Ca2+ concentration. Here we analyze the cytological effects and mode of action of the sulphydryl reagent thimerosal which could also trigger meiosis reinitiation in Ruditapes. No metaphase I spindle formed under these conditions since thimerosal was found to be able to preclude or reverse tubulin polymerization when applied to prophase- or to metaphase-arrested oocytes, respectively. Our results strongly suggest that the common final target for 5HT and thimerosal actions consists in a transient rise in internal free Ca2+ level that we could follow using Fluo3/AM as a probe. The effect of thimerosal in promoting oocyte maturation and increasing intracellular free Ca2+ concentration was improved by excess KCI. In addition, thimerosal, but not KCI, was found to facilitate 5HT-induced maturation at subthreshold hormone concentrations which, by themselves, did not produce an intracellular Ca2+ surge. These data suggest that thimerosal may inhibit Ca2+ pumps of the endoplasmic reticulum and unmask the plasma membrane voltage-sensitive Ca2+ channels which also appear after 5HT-induced GVBD.     

Hypertrophic cardiomyopathy. Role of Doppler echocardiography in the diagnosis and therapeutic approach

The present study was undertaken with the aim to study the role of isologous and heterologous (buffalo) oviductal cell to co-culture on in vitro development of goat embryos. The oocytes were collected by puncturing the goat ovaries obtained from slaughterhouse. The oocyte recovery rate per ovary was 3.0. The media used for oocyte maturation and embryo development was TCM-199 + 10 percent buffalo estrus serum. A total of 79.8 percent oocytes got matured out of 1056 oocytes. The oocytes were inseminated with epididymal buck spermatozoa capacitated in Brackett and Oliphant media. In group I without oviductal cells co-culture only 13.6 percent matured oocytes cleaved and 3.3 and 0.0 percent reached the morula and blastocyst stage. In group II and III having goat and buffalo oviductal cells the cleavage was 57.6 and 59.2 percent respectively. The percentage of morula, blastocyst and those embryos arrested between 2-16 cells were 26.3, 10.2, 63.5 and 26.6, 8.9 and 64.5 in goat and buffalo oviductal cell groups. The results indicated that the oviductal cell co-culture had a marked effect on cleavage and development of goat IVF embryos. Buffalo oviductal cells can be used well for goat embryo development.     

Antigens recognized by T-lymphocytes on human tumours

Some of the factors influencing the success of a nuclear transfer procedure are the quality of the recipient oocyte and the efficiency of the method of artificial activation. In this study we evaluated the ability of an electrical pulse to stimulate in vitro-matured porcine oocytes to develop. Maturation in Waymouth's medium resulted in significantly greater development than maturation in TCM-199 (p < 0.05), while there was no significant difference between degrees of development in Waymouth's medium and Whitten's medium. Oocytes matured in Waymouth's medium and electrically stimulated at 36 h (young oocytes) developed to the same degree as oocytes stimulated at 48 h (aged oocytes). Oocytes matured in Waymouth's medium and treated with cytochalasin B showed significantly greater development (p < 0.10) in response to electrical activation than controls. Staurosporine activation of oocytes resulted in significantly (p < 0.05) fewer morulae and blastocysts when compared to electrical stimulation. Development of parthenogenic embryos to the elongated filamentous stage (10% development beyond blastocyst) was obtained by maturing oocytes in Waymouth's medium and electrically stimulating them to develop. By obtaining development of porcine parthenotes beyond the blastocyst stage, we have identified an efficient method of oocyte maturation and oocyte activation for use in a system for nuclear transfer.     

Characterization of the embryotrophic activity of exogenous protein-free oviduct-conditioned medium used in culture of cattle embryos

Bovine zygotes, obtained after in vitro maturation and fertilization of oocytes from slaughtered cow ovaries, were cultured in droplets of nonconditioned or conditioned medium on bovine oviduct cell monolayers. The media tested were Medium 199 alone and Medium 199 supplemented with 10% fetal calf serum (FCS). Oviduct conditioning increased both early cleavage and development to blastocysts. Only the effect on early cleavage was mimicked by FCS. The blastocysts obtained in serum-free conditioned medium (SFCM) appeared morphologically normal and had the same cell number as those produced in conditioned medium containing serum. Their hatching rates did not differ. Transfer of 16 blastocysts developed in SFCM to 16 synchronized recipients resulted in five pregnancies (31%), indicating good embryonal viability. Boiling of SFCM resulted in a total loss of activity, while heating at 56 degrees C for 30 min had no deleterious effect. A 10-kDa ultrafiltration of SFCM removed the blastocyst development-supporting activity from the filtrate but not the early cleavage-favoring activity. This allows us to conclude that at least two different factors are present in SFCM: one of low molecular mass (< approximately 10 kDa), needed to obtain the 5-8 cell stage and mimicked by FCS, and another of higher molecular mass allowing embryos to develop from the 8-cell to blastocyst stage.     

Positive cooperativity of acetylcholine and other agonists with allosteric ligands on muscarinic acetylcholine receptors

At fertilization periodic Ca2+ oscillations release oocytes from meiotic arrest. The present study examined whether these oscillations have a long-term role in pre- and postimplantation development, independent of their immediate effect. Sr(2+)-containing medium was used to induce oscillations during exit from meiosis and first embryonic mitosis and Sr(2+)-activated parthenotes were compared to ethanol-activated parthenotes and embryos generated by in vitro fertilization. After embryo culture, blastocysts were differentially stained for the inner cell mass and trophectoderm. It was found that oscillations both during exit from meiosis and during mitosis acted to increase the number of inner cell mass cells. In contrast, the trophectoderm cell number was largest in ethanol-activated parthenotes and smallest in fertilized embryos. Postimplantation development was also modestly improved by extending the time of exposure to Sr(2+)-containing medium. Together these data suggest that Ca2+ oscillations have a role in long-term embryonic events and that they provide more than merely a stimulus for meiotic resumption.     

Succinate and malate improve development of hamster eight-cell embryos in vitro: confirmation of viability by embryo transfer

The in vitro development of hamster preimplantation embryos is supported by non-glucose energy substrates. To investigate the importance of embryonic metabolism, influence of succinate and malate on the development of hamster 8-cell embryos to blastocysts was examined using a chemically defined protein-free modified hamster embryo culture medium-2 (HECM-2m). There was a dose-dependent influence of succinate on blastocyst development; 0.5 mM succinate was optimal (85.1% +/- 3.9 vs. 54.5% +/- 3.5). In succinate-supplemented HECM-2m, blastocyst development was reduced by omission of lactate (68.5% +/- 7.2), but not pyruvate (85.8% +/- 6.2) or glutamine (84.1% +/- 2.1). Succinate along with either glutamine or lactate or pyruvate poorly supported blastocyst development (28%-58%). Malate also stimulated blastocyst development; 0.01 mM malate was optimal (86.3% +/- 2.8). Supplementation of both succinate and malate to HECM-2m supported maximal (100%) blastocyst development, which was inhibited 4-fold by the addition of glucose/phosphate. The mean cell numbers (MCN) of blastocysts cultured in succinate-supplemented HECM-2m was higher (28.3 +/- 1.1) than it was for those cultured in the absence of glutamine or pyruvate (range 20-24). The MCN was the highest (33.4 +/- 1.6) for blastocysts cultured in succinate-malate-supplemented HECM-2m followed by those in succinate (28.3 +/- 1.1) or malate (24.7 +/- 0.5) supplemented HECM-2m. Embryo transfer experiments showed that 29.8% (+/- 4.5) of transferred blastocysts cultured in succinate-malate-supplemented HECM-2m produced live births, similar (P > 0.1) to the control transfers of freshly recovered 8-cells (33.5% +/- 2.0) or blastocysts (28.9% +/- 3.0). These data show that supplementation of succinate and malate to HECM-2m supports 100% development of hamster 8-cell embryos to high quality viable blastocysts and that non-glucose oxidizable energy substrates are the most preferred components in hamster embryo culture medium.