Diffusion-weighted MR microscopy with fast spin-echo

A diffusion-weighted fast spin-echo (FSE) imaging sequence for high-field MR microscopy was developed and experimentally validated in a phantom and in a live rat. Pulsed diffusion gradients were executed before and after the initial 180 degrees pulse in the FSE pulse train. This produced diffusion-related reductions in image signal intensity corresponding to gradient ("b") factors between 1.80 and 1352 s/mm2. The degree of diffusion weighting was demonstrated to be independent of echo train length for experiments using trains up to 16 echoes long. Quantitative measurements on a phantom and on a live rat produced diffusion coefficients consistent with literature values. Importantly, the eight- to 16-fold increase in imaging efficiency with FSE was not accompanied by a significant loss of spatial resolution or contrast. This permits acquisition of in vivo three-dimensional data in time periods that are appropriate for evolving biological processes. The combination of accurate diffusion weighting and high spatial resolution provided by FSE makes the technique particularly useful for MR microscopy.     

Evidence for the requirement of autocrine growth factors for development of mouse preimplantation embryos in vitro

The mitotic stimuli in the early mammalian embryo have not been unequivocally identified. One hypothesis is that the embryo releases autocrine growth factors (GFs) that have a role in such growth. To determine whether such putative GFs were limited by dilution, and hence secreted, development was observed at various embryo concentrations in culture. Embryos were collected at the zygote or 2-cell stage. Zygotes were produced by fertilization in situ (ISF) or in vitro (IVF). Two-cell-stage embryos had a high rate of development to the blastocyst stage across an embryo concentration range of 1/microl-0.001/microl. By contrast, zygotes produced by either ISF or IVF were adversely affected by reducing the embryo concentration over this range (p < 0.001), with approximately 80% of ISF zygotes developing to blastocysts at the highest concentration but only 26% at the lowest. For IVF zygotes the corresponding results were 64% and 6%. For all three embryo types, the number of cells in each blastocyst was significantly lower with reduced embryo concentration. The major determinant of zygote development was the concentration of embryos in culture rather than the absolute volume of culture medium or the actual number of embryos present. A concentration of 1 embryo/microl (in the form of 10 embryos/10microl) gave the best development rates and highest cell numbers per blastocyst. Varying the albumin concentration influenced development rates; a 10-fold reduction in BSA concentration (to 0.3 mg/ml) resulted in significantly more IVF zygotes developing to the blastocyst stage. Platelet-activating factor (PAF) is released by embryos, and albumin can act as a competitive inhibitor of PAF's action on cells. ISF embryos released more PAF (p < 0.05) into media than did similarly treated IVF embryos. There was no difference in the amount of PAF remaining associated with the resulting 2-cell embryos. The amount of PAF released by both these groups was markedly less (p < 0.001) than the amount released by 2-cell embryos collected fresh from the reproductive tract and cultured for 24 h. PAF supplementation of media caused a significant increase in the rate of blastocyst development of IVF zygotes at embryo concentrations of 0.1/microl (1 ng/ml) and 0.01/microl (100 ng/ml). Insulin-like growth factor (IGF)-I (30 ng/ml) and IGF-II (1 ng/ml) also stimulated development of IVF zygotes when cultured at an embryo concentration of 1/10 microl. Epidermal growth factor was without effect over the range 0.2-2000 ng/ml. Supplementation of media with both PAF and IGF-II gave no additional benefit over that caused by IGF-II alone, but this treatment was marginally better (p < 0.05) than PAF treatment alone. The results show that factors necessary for normal embryo development are diluted to suboptimal levels during culture at low embryo concentration. The ability of PAF, IGF-I, and IGF-II to partially compensate for the adverse effects of low embryo concentration during culture is consistent with their having roles as autocrine embryotrophic factors. The use of IVF and low embryo concentrations in culture may provide a functional multiple ablation model that will help to define the range of GFs required for normal embryo development.     

Development of in vitro derived bovine embryos following pronuclear transplantation and in vitro culture

This study was designed to evaluate the survival and development of in vitro derived bovine embryos following pronuclear transplantation and in vitro embryo culture. Bovine zygotes were produced by in vitro maturation and in vitro fertilization. Pronuclei were removed by micromanipulation and either transferred back to the same cell (Group 1) or into a previously enucleated zygote (Group 2) by electrofusion. Micromanipulated and non-micromanipulated (Group 3, control) zygotes were co-cultured with oviductal cells in a sealed modular chamber filled with 5% CO2, 5% O2 and 90% N2 at 39 degrees C for 7-8 days. Fusion rates were similar for Groups 1 and 2 (90.7 and 85.1%, respectively, P > 0.05). The percentage of embryos that cleaved was not different for Groups 1 (82.0%), 2 (90.0%) and 3 (76.9%, P > 0.05). Also, the percentage of embryos developing to the compact morula or blastocyst stage was similar (25.6, 22.5 and 22.3%, respectively, for Groups 1, 2 and 3, P > 0.05). The results of this experiment are the first to demonstrate that pronuclear transfer can be carried out successfully using bovine embryos derived from in vitro oocyte maturation and in vitro fertilization. In addition, pronuclei can be transferred from one bovine embryo to another and the reconstructed embryos develop to the compact morula and blastocyst stage in vitro. This technique, used in combination with oocyte retrieval by ultrasound-guided follicular aspiration and embryo transfer, offers the potential to study cytoplasmic inheritance in cattle directly, and to evaluate the effect of cytoplasmic inheritance on traits of economic importance.     

Functional analysis of a rat sodium channel carrying a mutation for insect knock-down resistance (kdr) to pyrethroids

As a model for establishing an optimized medium for human in vitro fertilization (IVF), modified human tubal fluid (HTF) media containing amino acids at concentrations found in human serum and follicular fluid were prepared, and the effect of the media on development of random-bred (ICR) and F1 hybrid (CBF1) mice embryos was studied. The total concentrations of amino acids found in serum and follicular fluid were about one-third to one-half the concentrations present in two conventional media used in human IVF: Ham's F-10 and Eagle's minimal essential medium (MEM). When ICR mouse embryos were cultured in the HTF medium containing 21 amino acids at concentrations found in follicular fluid, the number of embryos developing to morulae at 72 h and to blastocysts at 96 h increased in comparison with those cultured in HTF medium. When HTF containing amino acids at concentrations found in serum was used, only induced morula formation at 72 h was enhanced. The number of hatching blastocysts at 96 h also increased when CBF1 mouse embryos were cultured with HTF supplemented with amino acids at concentrations found in follicular fluid. When ICR mouse embryos were cultured in modified HTF media containing concentrations of amino acids found in Ham's F-10 and MEM that contained higher concentrations of glutamine, embryo development was inhibited. The amount of ammonium produced during incubation for 3 days was significantly less when embryos were cultured in media containing concentrations of amino acids found in follicular fluid compared with when Ham's F-10 or MEM was the culture medium. Ammonium is produced by the breakdown of glutamine in the culture medium during incubation with or without embryos. These results suggest that the concentrations of amino acids found in follicular fluid are more effective and safer for embryo culture than those in other media currently in use.     

Electron microscopic study of the rat submaxillary gland during development

Cytological changes in the submaxillary glands of rat embryos of the 14th-21st days of the intrauterine development and of 7-day-old rats were studied electron microscopically. The cytoplasm of the salivary gland primordium cells of the 14-day embryo was found to possess multiple polysomes, the canals of the granular cytoplasmic network (GCN) being solitary. The latter elongated in the process of embryonic development, and later predominant development of GCN was observed in basal portions of the acinar cells. At early stages of the gland development the GCN was often observed to approximate mitochindria. The laminar complex in the course of embryonic development enlarges its size and at later stages it has a well differentiated appearance in the acinar cells. Secretory inclusions make their appearance in the forming cells of acinuses long before birth, the composition of the embryo gland secretion being somewhat different from that of adult animals. Differentiation of acinuses and striated parts of the salivary tubules is well pronounced in the embryonic period while no signs of appearance of granular portions of the salivary tubules are observed both in light and electron microscopy either in embryos or in 1-week-old rats.     

Cell-cell association directed mitotic spindle orientation in the early development of the marine shrimp Sicyonia ingentis

During early cleavages of Sicyonia ingentis embryos, mitotic spindle orientations differ between blastomeres and change in a predictable manner with each successive mitosis. From 2nd through 7th cleavages, spindles orient at a 90 degrees angle with respect to the spindle of the parent blastomere. Thus, spindle orientation is parallel to the cleavage plane that formed the blastomere. To determine if specific spindle orientations were intrinsic properties of individual blastomeres, we altered blastomere associations and asked how mitotic spindle orientation was affected in successive cleavages using laser scanning confocal microscopy. Linear embryos were constructed by dissociating 4-cell embryos and recombining the blastomeres in a linear array. The ensuing cleavage (3rd embryonic cleavage) of these linear embryos was parallel to the long axis of the embryo, resulting in four parallel pairs of blastomeres which lay in a common plane that was parallel to the substratum. The 4th cleavage produced a linear embryo with the 16 blastomeres arranged in four parallel quartets. Then, in preparation for 5th cleavage, spindles oriented at a 45 degrees angle (not parallel as in normal development) with respect to the previous cleavage plane. When 8-cell linear embryos were separated into linear half-embryos, subsequent spindle orientations were not like those observed for intact 8-cell linear embryos, but rather regressed to the orientation seen in 4-cell linear embryos. We suggest that the reorientation of mitotic spindles during early cleavage of S. ingentis is neither an intrinsic property nor age dependent, but rather is cell contact related. Further, these results in conjunction with observations of non-manipulated embryos suggest that spindle poles (centrosomes) avoid cytoplasmic regions adjacent to where there is cell-cell contact during early development.     

Developmental spectrum of cardiac outflow tract anomalies encompassing transposition of the great arteries and dextroposition of the aorta: pathogenic effect of extrinsic retinoic acid in the mouse embryo

We previously reported that retinoic acid shows a dose-dependent differential induction of various cardiac outflow anomalies: transposition of the great arteries is induced mainly by a high dose (70 mg/kg) and dextroposition of the aorta by a low dose (40-60 mg/kg; Yasui et al., 1995). We subsequently delineated the aberrant outflow tract septation process leading to the transposition (Yasui et al., 1997). The aim of the present study was to illustrate a spectrum of developmental abnormalities by examining mouse embryos treated with a low dose of retinoic acid and comparing them with embryos administered a high dose. We employed in situ observation on live embryos to discern the blood flow streams and scanning electron microscopy to clarify the internal structure. The embryos treated with a low dose of retinoic acid showed several basic phenotypes common to the high dose retinoic acid group, although variable and relatively mild, such as hypoplasia and dysplasia in the proximal outflow cushions, decreased counter-clockwise rotation in the distal outflow tract, and deviation of the edges of the developing outflow septum. In typical cases, the right-sided edge of the developing outflow septum shifted ventrally by various degrees, allowing for the right ventricle-to-aorta pathway, whereas the left-sided edge preserved the continuity with the interventricular septum, as in the normal embryo. These findings indicate that morphogenesis of dextroposition of the aorta and transposition of the great arteries are not only distinct but also show some basic pathways in common.     

Current and future methodology for monitoring sleep

The present study examined the effect of follicular shell pieces (FSP) during in vitro maturation (IVM) of porcine oocytes on 1) in vitro fertilization (IVF) parameters, 2) subsequent embryo development, 3) oocyte glutathione (GSH) concentration, and 4) viability after embryo transfer. Cumulus-oocyte complexes were cultured in North Carolina State University (NCSU) 23 medium containing porcine follicular fluid, cysteine, and hormonal supplements and with or without FSP for 20-22 h. They were then cultured in the same medium but without hormonal supplements for an additional 20-22 h. After culture, cumulus-free oocytes were coincubated with frozen-thawed spermatozoa for 5-6 h. Putative zygotes were transferred to NCSU 23 containing 0.4% BSA and cultured for 144 h. In comparisons between the presence and absence of FSP, no differences were observed in fertilization parameters. At 48 h, no mean differences were found in cleavage rates. However, at 144 h, the proportion of embryos that developed to the blastocyst stage was significantly (p < 0.01) higher (18% vs. 36%) for oocytes cocultured with FSP. A significantly (p < 0.05) higher GSH concentration was found in oocytes matured with FSP as determined by dithionitrobenzoic acid-glutathione disulfide (DTNB-GSSG) reductase recycling assay. Transfer of embryos to 9 recipients resulted in 5 pregnancies with the birth of 18 live piglets. The results provide clear evidence of the beneficial effect of FSP during IVM of pig oocytes cultured in the presence of cysteine on subsequent embryo development to the blastocyst stage. The birth of piglets confirms the viability of IVM-IVF-derived embryos.     

Developmental potential of embryos produced by in-vitro fertilization from gonadotrophin-releasing hormone antagonist-treated macaques stimulated with recombinant human follicle stimulating hormone alone or in combination with luteinizing hormone

We previously demonstrated, in luteinizing hormone (LH)-deficient macaques, that follicular growth and maturation occurred with administration of exogenous (recombinant human) follicle stimulating hormone (r-hFSH) alone, and that the oocytes recovered fertilized at a notably higher rate than their counterparts from animals receiving both r-hFSH and r-hLH (Zelinski-Wooten et al., 1995). Here, the developmental potential of embryos produced from animals treated with r-hFSH alone or in combination with r-hLH was evaluated. Embryos (n = 127) were cryopreserved, thawed and either co-cultured on buffalo rat liver cells until the hatched blastocyst stage or transferred to synchronized recipients. Although embryos from each treatment group demonstrated a similar ability to develop to hatched blastocysts with a definitive inner cell mass, a significant difference was seen in cryosurvival (56 versus 78%) and in developmental rate to the hatched blastocyst (12 versus 10 days) between embryos from the r-hFSH alone and the combination group respectively. Pregnancies resulted following oviductal embryo transfers in both groups, with corpus luteum rescue occurring on days 12-16 of the luteal phase. In summary, r-hFSH alone during the pre-ovulatory interval is adequate for the gametogenic events required to produce embryos that develop either in vitro or in vivo; however, exposure to r-hLH may improve embryo viability and the rate of development.     

Magnetic resonance microscopy and histopathology: comparative approach of bromobenzene-induced hepatotoxicity in the rat

The development of magnetic resonance (MR) microscopy has provided new approaches to histology and histopathology. Recent work has shown the promise of increased sensitivity in animal models of chemically induced hepatotoxicity. However, the field is so new that there is little experience to relate changes seen in MR micrographs to the more traditional optical images stained with hematoxylin and eosin. This work compares the sensitivity and reproducibility of MR microscopy with conventional histopathology in detecting bromobenzene-induced hepatotoxicity in the rat. A time-course study was undertaken to provide a range of histopathologies. Specimens were studied at 24, 48, 72, and 96 hours after exposure to 10% of the median lethal dose of bromobenzene. Using 4 animals per group (a total of 32 rats) added statistical significance to the study and defined a range of interanimal variability over 96 hours. This work shows that MR microscopy, besides being nondestructive and three-dimensional, is at least as sensitive as conventional hematoxylin-eosin staining in detecting bromobenzene-induced centrilobular lesions and recovery of the hepatocellular architecture in the rat. This study further suggests that, as we begin to understand the underlying mechanisms of contrast in MR histology, MR may, in fact, supply even higher specificity than more traditional studies: variations were observed in MR images of treated livers at a given time point that could be not be differentiated based on the grading of necrosis and inflammation on hematoxylin-eosin-stained sections.     

Erythroid carbonic anhydrases of developing chick embryos. Coordinate expression with primitive and definitive embryonic haemoglobins

Erythroid carbonic anhydrase activity of chick embryos from the 3rd day of incubation to the egg hatching has been determined. Three minor activity peaks at 3, 9 and 15 days of development and a major one at 19 days were found. The enzyme molecular forms were purified by affinity chromatography from haemolysates of embryos at several stages of development. As has been found for the adult erythrocytes, only type II isozyme was detected in the embryo red cells. Isoelectrofocusing analysis demonstrated that two different molecular forms of this isozyme are synthesized by the red cells of developing embryos. Only the early form is present up to 5 days of development; the late form, which is indistinguishable from the adult isozyme, appears in the haemolysate at 6-7 days and quickly replaces the early form. Analysis of purified primitive and definitive erythroid lines from 7-days-old embryos showed a compartmentalization of the early and late forms into the primitive and definitive erythroid cells, respectively.     

Method for treating and processing whole chick embryos for autoradiography, immunocytochemistry and other techniques

A method is presented for in situ treatment of whole chick embryos with drugs and immunocytochemical and fixative reagents that resembles conditions "in ovo." The chick embryo is placed in a "shell-less" culture system where it is contained by an agar ring allowing for treatment in vivo. The conceptus (embryo+membranes) is then mounted on a microporous membrane and inserted into a filter device connected to a three-way stopcock that permits fluids to be changed using syringes. The embryo is then processed in toto or after embedding and sectioning for light or electron microscopy. The proposed handling system decreases technical artifacts and changes in the topographic microanatomy produced by conventional manipulation of chick embryos. This method is useful also for directly observing and recording changes in the embryo during drug treatments and allows processing with dangerous reagents without their direct contact with the operator. It is simple, inexpensive and requires only minimal technical training.     

Progressive effect of alpha-phenyl-N-tert-butyl nitrone (PBN) on rat embryo development in vitro

In the present study we demonstrated the effects of the spin-trapping agent alpha-phenyl-N-tert-butylnitrone (PBN) on the in vitro development of rat embryos at the early stage. In rat embryos, PBN increased the speed of the first cleavage and had no toxicity during pregnancy after embryo culture. These results showed that reactive oxygen species (ROIs) that were formed by activating molecular oxygens through redox reactions regulated the speed of development for early-stage embryos. Thus, PBN caused a decrease in the level of ROIs and toxicity and an in increase in the level of the development of rat embryos. On the other hand, PBN could not decrease the 2-cell block in vitro nor increase the blastulation rate, in contrast to the fact that a scavenger of superoxide anions, SOD, is effective in doing so for mouse embryos. From these results it was concluded that free radicals play an important role in the in vitro development of rat embryos at the early stage, but play no role in the decrease of the 2-cell block or their blastulation rate. It should be noted that PBN had no toxicity for embryonic development at the 2-cell stage.     

Chromosome region 8p11-p21: refined mapping and molecular alterations in breast cancer

This review illustrates the use of experimental approaches combined with microscopy to study the biology of monogenean parasites. Studies of feeding, development, reproduction, and systematics have been based on gyrodactylids, flatworms infecting teleost fishes. In a contrasting system involving an amphibian host in a desert environment, analysis of adaptations to extreme conditions has focused on Pseudodiplorchis americanus. The unusual reproductive strategies, particularly the interactions between mother and offspring, are highlighted for both monogeneans. Species of Gyrodactylus are viviparous, maintaining up to three generations of embryos simultaneously in utero, and many of their reproductive specializations are related to progenesis. Embryo nutrition takes place via a metabolically-active syncytial uterine lining that has close association with the parental gut. Microscopy has also proved an essential adjunct to molecular studies of speciation and host specificity. P. americanus is ovoviviparous and the adaptations for embryo maintenance are unique. The primary keratin-type eggshell is replaced by a flexible secondary elastin capsule produced by the uterus; parental nutrients are transferred through cytoplasmic connections to the developing embryo. TEM has demonstrated unique adaptations of P. americanus to its micro-environments, including secretion of tegumental vesicles that provide protection from digestive enzymes during migration through the host gut. This paper highlights the potential of monogeneans for studies of fundamental biological principles.     

Blunt impact causes changes in bone and cartilage in a regularly exercised animal model

Confocal laser scanning microscopy combined with a vital stain has been used to study apoptosis in organogenesis-stage mouse embryos. In order to achieve optical sectioning through embryos, it was necessary to use low power objectives and to prepare the sample appropriately. Mouse embryos were harvested on gestation day 8 or 9 and stained with the vital lysosomal dye, LysoTracker Red. Following incubation in the stain, embryos were fixed in 2% paraformaldehyde overnight, dehydrated in a graded methanol series, and cleared in benzyl alcohol/benzyl benzoate. The resulting embryo is almost transparent and retains specific LysoTracker Red staining. The entire embryo can be optically sectioned and reconstructed in three dimensions to reveal areas of dye staining. To test this approach, the chemotherapeutic drug hydroxyurea was added to day 8 embryos in vitro to induce apoptosis. Our results demonstrated specific regions undergoing programmed cell death in normal development and increased apoptosis in embryos exposed to hydroxyurea. The observed patterns of LysoTracker Red staining correlate well with previous studies of cell death using other lysosomotropic dyes such as Nile blue sulfate, acridine orange, or neutral red. LysoTracker Red has the advantages of being aldehyde-fixable and highly fluorescent (bleaching was not observed even after multiple scans). This procedure allows for the optical imaging of whole day 9 (approximately 22 somites) embryos that were greater than 500 microns thick in the Z-axis.     

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.     

Comparison of monolayer and bilayer plates used in antibiotic assay

Development of the rat embryo is arrested at the 2-cell stage in vitro in the presence of inorganic phosphate (Pi). Rat embryos were affected by exposure to 1.19 mM KH2PO4 in modified hamster embryo culture medium-1 at the late 2-cell stage only. When exposure durations were 6 h, embryos whose exposure timings were prior to cleavage had a reduced rate of development to the blastocyst stage (2-8%) when compared with embryos with no exposure to Pi (97%, P < 0.05). When exposure durations were 18 h, all embryos were arrested at the 2- to 4-cell stage. These timings would correspond to the G2 to M phase of the second cell cycle. Maturation-promoting factor (MPF), which is regulated by a phosphorylation cascade, controls cell division, and its kinase activity is necessary in order for the cell to enter the M phase. However, the histone H1 kinase activity levels and the patterns of the state of phosphorylation of cdc2 were the same in blocked and non-blocked embryos. Because MPF was active in blocked embryos, the developmental block in rat 2-cell embryos caused by phosphate was not due to MPF activity or its phosphorylation cascade.     

Differences on post-thawing survival between ovine morulae and blastocysts cryopreserved with ethylene glycol or glycerol

Embryos were collected on Days 5 and 6 after breeding to investigate the effectiveness of ethylene glycol (ETG) and glycerol (GLY) as cryoprotectants of sheep morulae and blastocysts and to determine their optimum stage of development for cryopreservation. Only excellent (grade 1) and good (grade 2) embryos (196 morulae and 188 blastocysts) were incubated in increasing concentrations of GLY or ETG and submitted to a slow-freezing and quick-thawing procedure. Both cryoprotectants were removed using 0.25 M sucrose solution, and then embryos were cultured or transferred to determine their viability. Freezing medium containing ETG yielded higher in vitro survival rates (P < 0.01) than medium containing GLY (64.6% vs 16.0%); the difference between cryoprotectants was greater when morulae were used (57.9% vs 4.2%, P < 0.005) as compared with blastocysts (70.4% vs 21.5%, P < 0.05). There was a strong interaction between type of cryoprotectant and embryo stage (P < 0.005). After transfer of morphologically viable embryos, the in vivo development rate of embryos frozen with ETG was also higher than that of embryos frozen with GLY (45.5% vs 27.7%, P < 0.05). There were no significant differences in the number of lambs born among procedures and embryo stage, though the lowest lambing rate was obtained with morulae frozen with GLY (21.4%). Similar lambing rates were produced when blastocysts were frozen with either GLY or ETG (36.6% vs 43.0%). The best embryo survival after thawing was observed when blastocysts were frozen with ETG as cryoprotectant.