Water in the terrestrial planet-forming zone of the PDS 70 disk.

Terrestrial and sub-Neptune planets are expected to form in the inner (less than 10 AU) regions of protoplanetary disks1. Water plays a key role in their formation2-4, although it is yet unclear whether water molecules are formed in situ or transported from the outer disk5,6. So far Spitzer Space Telescope observations have only provided water luminosity upper limits for dust-depleted inner disks7, similar to PDS 70, the first system with direct confirmation of protoplanet presence8,9. Here we report JWST observations of PDS 70, a benchmark target to search for water in a disk hosting a large (approximately 54 AU) planet-carved gap separating an inner and outer disk10,11. Our findings show water in the inner disk of PDS 70. This implies that potential terrestrial planets forming therein have access to a water reservoir. The column densities of water vapour suggest in-situ formation via a reaction sequence involving O, H2 and/or OH, and survival through water self-shielding5. This is also supported by the presence of CO2 emission, another molecule sensitive to ultraviolet photodissociation. Dust shielding, and replenishment of both gas and small dust from the outer disk, may also play a role in sustaining the water reservoir12. Our observations also reveal a strong variability of the mid-infrared spectral energy distribution, pointing to a change of inner disk geometry.

A dusty compact object bridging galaxies and quasars at cosmic dawn.

Understanding how super-massive black holes form and grow in the early Universe has become a major challenge1,2 since it was discovered that luminous quasars existed only 700 million years after the Big Bang3,4. Simulations indicate an evolutionary sequence of dust-reddened quasars emerging from heavily dust-obscured starbursts that then transition to unobscured luminous quasars by expelling gas and dust5. Although the last phase has been identified out to a redshift of 7.6 (ref. 6), a transitioning quasar has not been found at similar redshifts owing to their faintness at optical and near-infrared wavelengths. Here we report observations of an ultraviolet compact object, GNz7q, associated with a dust-enshrouded starburst at a redshift of 7.1899 ± 0.0005. The host galaxy is more luminous in dust emission than any other known object at this epoch, forming 1,600 solar masses of stars per year within a central radius of 480 parsec. A red point source in the far-ultraviolet is identified in deep, high-resolution imaging and slitless spectroscopy. GNz7q is extremely faint in X-rays, which indicates the emergence of a uniquely ultraviolet compact star-forming region or a Compton-thick super-Eddington black-hole accretion disk at the dusty starburst core. In the latter case, the observed properties are consistent with predictions from cosmological simulations7 and suggest that GNz7q is an antecedent to unobscured luminous quasars at later epochs.

A massive core for a cluster of galaxies at a redshift of 4.3.

Massive galaxy clusters have been found that date to times as early as three billion years after the Big Bang, containing stars that formed at even earlier epochs1-3. The high-redshift progenitors of these galaxy clusters-termed 'protoclusters'-can be identified in cosmological simulations that have the highest overdensities (greater-than-average densities) of dark matter4-6. Protoclusters are expected to contain extremely massive galaxies that can be observed as luminous starbursts 7 . However, recent detections of possible protoclusters hosting such starbursts8-11 do not support the kind of rapid cluster-core formation expected from simulations 12 : the structures observed contain only a handful of starbursting galaxies spread throughout a broad region, with poor evidence for eventual collapse into a protocluster. Here we report observations of carbon monoxide and ionized carbon emission from the source SPT2349-56. We find that this source consists of at least 14 gas-rich galaxies, all lying at redshifts of 4.31. We demonstrate that each of these galaxies is forming stars between 50 and 1,000 times more quickly than our own Milky Way, and that all are located within a projected region that is only around 130 kiloparsecs in diameter. This galaxy surface density is more than ten times the average blank-field value (integrated over all redshifts), and more than 1,000 times the average field volume density. The velocity dispersion (approximately 410 kilometres per second) of these galaxies and the enormous gas and star-formation densities suggest that this system represents the core of a cluster of galaxies that was already at an advanced stage of formation when the Universe was only 1.4 billion years old. A comparison with other known protoclusters at high redshifts shows that SPT2349-56 could be building one of the most massive structures in the Universe today.

Author Correction: A massive core for a cluster of galaxies at a redshift of 4.3.

Change history: In this Letter, the Acknowledgements section should have included the following sentence: "The National Radio Astronomy Observatory is a facility of the National Science Foundation operated under cooperative agreement by Associated Universities, Inc.". This omission has been corrected online.

Galaxy growth in a massive halo in the first billion years of cosmic history.

According to the current understanding of cosmic structure formation, the precursors of the most massive structures in the Universe began to form shortly after the Big Bang, in regions corresponding to the largest fluctuations in the cosmic density field. Observing these structures during their period of active growth and assembly-the first few hundred million years of the Universe-is challenging because it requires surveys that are sensitive enough to detect the distant galaxies that act as signposts for these structures and wide enough to capture the rarest objects. As a result, very few such objects have been detected so far. Here we report observations of a far-infrared-luminous object at redshift 6.900 (less than 800 million years after the Big Bang) that was discovered in a wide-field survey. High-resolution imaging shows it to be a pair of extremely massive star-forming galaxies. The larger is forming stars at a rate of 2,900 solar masses per year, contains 270 billion solar masses of gas and 2.5 billion solar masses of dust, and is more massive than any other known object at a redshift of more than 6. Its rapid star formation is probably triggered by its companion galaxy at a projected separation of 8 kiloparsecs. This merging companion hosts 35 billion solar masses of stars and has a star-formation rate of 540 solar masses per year, but has an order of magnitude less gas and dust than its neighbour and physical conditions akin to those observed in lower-metallicity galaxies in the nearby Universe. These objects suggest the presence of a dark-matter halo with a mass of more than 100 billion solar masses, making it among the rarest dark-matter haloes that should exist in the Universe at this epoch.

Dusty starburst galaxies in the early Universe as revealed by gravitational lensing.

In the past decade, our understanding of galaxy evolution has been revolutionized by the discovery that luminous, dusty starburst galaxies were 1,000 times more abundant in the early Universe than at present. It has, however, been difficult to measure the complete redshift distribution of these objects, especially at the highest redshifts (z > 4). Here we report a redshift survey at a wavelength of three millimetres, targeting carbon monoxide line emission from the star-forming molecular gas in the direction of extraordinarily bright millimetre-wave-selected sources. High-resolution imaging demonstrates that these sources are strongly gravitationally lensed by foreground galaxies. We detect spectral lines in 23 out of 26 sources and multiple lines in 12 of those 23 sources, from which we obtain robust, unambiguous redshifts. At least 10 of the sources are found to lie at z > 4, indicating that the fraction of dusty starburst galaxies at high redshifts is greater than previously thought. Models of lens geometries in the sample indicate that the background objects are ultra-luminous infrared galaxies, powered by extreme bursts of star formation.

A simple method to quantify follicle survival in cryopreserved human ovarian tissue.

STUDY QUESTION: Can follicle survival in frozen-thawed human ovarian tissue be quantified in situ using the dye Neutral Red (NR) to stain viable follicles specifically? SUMMARY ANSWER: A follicle survival rate within ovarian tissue can be calculated using NR followed by histological evaluation and evidence for a consistently high follicle survival in a series of ovarian tissue from 25 Danish girls and women undergoing ovarian tissue cryopreservation (OTC) was obtained. WHAT IS KNOWN ALREADY: Securing follicle survival in cryopreserved ovarian tissue is crucial for proper quality control when centers wish to implement OTC. The only established technique for validation of follicle survival is xenografting of thawed ovarian tissue to immunodeficient mice. However, this functional test is expensive, time consuming, requires animal facilities and only provides a qualitative-not quantitative-measure for follicle survival. STUDY DESIGN SIZE, DURATION: Quantification of follicle survival in human ovarian tissue donated from 30 girls and women having tissue cryopreserved for fertility preservation from 2000 to 2015 at the Laboratory of Reproductive Biology in Copenhagen, Denmark. PARTICIPANTS/MATERIALS, SETTING, METHODS: Cryopreserved ovarian cortex was donated from 25 girls and young women aged 10-36 years (mean age: 25 years) and the average storage time in liquid nitrogen was 9.1 ± 5.6 years, ranging from 1.6 to 17.9 years. In 12 of the cases, the ovarian tissue was collected from the local hospital and in the other 13 cases the ovarian tissue was transported on ice up to 6 h prior to freezing. Donated fresh ovarian surplus tissue was obtained from five women aged 23-34 years (mean age: 27 years). Ovarian tissues were chopped into small fragments and incubated in culture medium containing 50 mg/ml NR for 3-4 h. Fragments of ovarian tissue containing clearly NR-stained follicles were selected for counting, encapsulated in 4% agar and were processed for histology to calculate a follicular survival rate. MAIN RESULTS AND THE ROLE OF CHANCE: The mean follicle survival rate in the 25 patients after freezing and thawing was 84% ± 11 (mean ±SD), ranging from 50% to 98%. The high follicle survival rate in this clinical series of patients reflects a constant high-quality service performed in our center and confirms the robustness of the slow freezing protocol. No significant association between follicle survival rates and storage time was found using linear regression analysis, suggesting that storage in liquid nitrogen does not affect viability of the tissue. No significant association in follicle survival rates was found between ovarian tissues collected at the local hospital compared to tissues transported on ice prior to freezing, supporting that prolonged cooling does not seem to greatly affect the follicle survival. For the fresh ovarian tissue, the average follicle survival rate was 91% ± 5 (mean ± SD) in five patients, ranging from 81% to 95%. LIMITATIONS, REASONS FOR CAUTION: Even though the NR staining requires active incorporation of the dye, the test is merely a short in situ test that cannot completely replace the functional value of xenografting studies in which the integrity and developmental potential of the ovarian follicles are assessed. WIDER IMPLICATIONS OF THE FINDINGS: OTC is now being employed around the world but to date it has been difficult for centers to evaluate the effectiveness of their program and perform proper quality control. NR staining combined with histological evaluation is the first quantitative method to provide a survival rate for follicles in frozen-thawed human ovarian tissue and offer a valuable and easily applicable tool to validate the cryopreservation procedure when implementing OTC or as routine quality control for the overall freezing performance within tissue banking facilities. STUDY FUNDING/COMPETING INTEREST(S): The Research Pools of Rigshospitalet, the Danish Cancer Foundation, Dagmar Marshalls Foundation, and the Novo Nordic Foundation are thanked for having funded this study. The authors have no conflicts of interest.

Fertility in cancer patients after cryopreservation of one ovary.

This questionnaire study describes the fertility and ovarian function in 143 adult female cancer survivors with only one ovary due to cryopreservation of the other. The women were asked about their ovarian function (as defined by the presence of a spontaneous menstrual cycle), pregnancies and their outcome. The mean follow-up time was 58months after cryopreservation (range 24-129months). The risk of premature ovarian failure was high in the group of patients with leukaemia (13/15; 87%) but low in the breast cancer group (5/54; 9%). Fifty-seven women had actively tried to become pregnant after end of treatment; of these, 41 women obtained a total of 68 pregnancies resulting in 45 live births and five ongoing pregnancies, 15 spontaneous abortions, one ectopic pregnancy and two elective abortions. In the remaining 86 women without a pregnancy wish, there had been five elective abortions. Ninety-three per cent of the pregnancies were after natural conception and only four cases were a result of fertility treatment. The overall risk of premature ovarian failure was low (22%). Patients who retain their ovarian function after treatment of a malignant disease have a good chance of becoming pregnant.

Respiration rates correlate with mRNA expression of G6PD and GLUT1 genes in individual bovine in vitro-produced blastocysts.

Quantification of embryo respiration is a promising procedure to assess embryonic metabolism and possibly select viable embryos. At the blastocyst stage, ATP is produced by glycolysis and oxidative phosphorylation, processes that require uptake of oxygen and glucose, which is regulated by the expression of GLUT1 and G6PD. The purpose of the present study was to investigate the relationship between respiration rates and relative abundances of G6PD and GLUT1 transcripts in individual bovine blastocysts produced in vitro. Respiration rates of 104 bovine in vitro-produced blastocysts were measured individually using the nanorespirometer technology. Real-time RT-PCR was employed to determine the relative abundance of G6PD and GLUT1 mRNA in individual embryos. The mean respiration rates were similar for male and female blastocysts of the same developmental stage, but the sex ratio was skewed towards males. GLUT1 expression was down-regulated in female versus male embryos. In contrast, a approximately 1.8-fold increase in the expression of G6PD mRNA was observed in female blastocysts when compared to male blastocysts, indicating that dosage compensation for this gene had not yet occurred. Both GLUT1 and G6PD expression levels were affected by morphological quality and stage of development. Expression of GLUT1 and G6PD mRNAs was correlated with respiration rates, indicating that, in metabolically active blastocysts, uptake of oxygen and glucose are jointly increased. These findings suggest that expression of genes for oxidative phosphorylation and glycolysis are both involved in oxygen demanding ATP production.

Embryo technology: implications for fertility in cattle.

During the past thirty years, basic and experimental studies on classical (superovulation; non-surgical recovery and transfer of cattle embryos) and advanced embryo technologies (in vitro embryo production; cloning by somatic cell nuclear transfer) have generated structural and functional information on oocyte development and quality, fertilisation and conceptus development. This information has provided new insight, not only into these technologies per se but also into the factors contributing to fertility in cattle. It is now known that the peripheral and follicular endocrine profiles have a profound influence on the subsequent developmental competence of the embryo. It is also well established that manipulation of the oocytes or embryos may adversely affect embryonic and foetal development, leading to the so-called 'large offspring syndrome'. Information from such studies has alerted scientists to the importance of epigenetics in cattle reproduction.

Morphology of the oocyte-follicular connection in the mare.

The present study characterised the oocyte-follicular connection (i.e., oocyte fixation site) in Graafian follicles of the mare morphologically. Antral follicles were dissected in toto from ovaries obtained from oestrous, dioestrous and transitional mares after slaughter. The location of the cumulus oophorus complex in relation to the ovulation fossa, the width and density of the blood vessels surrounding the cumulus oophorus complex, the relative dimensions and histological aspects of the cumulus oophorus were investigated. For ultrastructural analysis of the junctional regions, cumulus-oocyte complexes were recovered in vivo by transvaginal ultrasound-guided follicle aspiration. The location of the oocyte fixation site was independent of mare, follicular size and stage of the oestrous cycle. In 82% of follicles, the oocytes were embedded in a broad based cell mount. The width and density of the blood vessels surrounding the oocyte fixation site were correlated to each other, but independent of follicular size and cyclic stage. The histological appearance of the cumulus oophorus varied, especially in respect to the compactness, and loosening of the cumulus cell population was observed in several medium-sized follicles from dioestrous mares. Loosening of the cumulus cell population was apparently associated with decreased interdigitation between adjacent corona radiata cells. It can be concluded that the fixation site of the equine cumulus oophorus complex represents a firm cellular anchorage between follicular wall and oocyte. Furthermore, the location of the cumulus oophorus complex in relation to the ovulation fossa and characteristics of the surrounding blood vessels is independent of follicular size and cyclic stage.

A comparative ultrastructural study of in vivo versus in vitro fertilization of bovine oocytes.

Heifers were superovulated by PMSG or FSH, and oestrus was induced by prostaglandin. One group of animals was ovariectomized 19-26 h after the LH peak, the content of preovulatory follicles aspirated, and the oocytes processed for in vitro fertilization. Another group was inseminated and ova were collected from the oviducts for study of in vivo fertilization. All ova were examined ultrastructurally. The developmental rate following in vitro fertilization was delayed compared to fertilization in vivo. A high proportion of the in vitro fertilized ova showed polyspermic penetration of the zona pellucida, and supernumerary spermatozoa were found in the ooplasm of some ova. In vivo fertilization was associated with release and subsequent dispersal of the cortical granule content in the perivitelline space. In contrast to this the released granule content of the in vitro fertilized ova remained undispersed close to the oolemma. This feature may account for the high incidence of polyspermic penetration of the zona pellucida. In addition, the study provided an ultrastructural visualization of the initial contact between the equatorial segment of the spermatozoon and the microvilli of the oocyte, and the subsequent internalization of the sperm head.

Ultrastructural features of secretory cells in the bovine oviduct epithelium.

The non-ciliated (NC) cells of the bovine oviduct epithelium, have been shown to release embryotrophic substances to the oviduct lumen. The aim of the present study was to investigate the ultrastructure, focusing on aspects of the secretory machinery, of NC cells in different segments of the oviduct during and after transoviduct migration of zygotes and embryos. Dairy heifers (n = 8) were superovulated with an ECG/cloprostenol regimen, and the time of ovulation was estimated by ultrasound scanning. Samples from the infundibulum, ampulla, isthmus and uterotubal-junction of the oviduct were surgically collected from animals at 19-96 h and 7 1/2-8 1/2 days after ovulation and processed for transmission electron microscopy, following standard procedures. The NC cells contained characteristic membrane-bound secretory granules composed of a lamellar cortex encaging an amorphous medulla. The two components could still be recognized during extrusion of the granule content into the oviduct lumen by exocytosis. During granulogenesis, small maturing granules without the lamellar structure were observed, but distinct condensing vacuoles were absent. An abundance of granules was found in the early versus the late group. In both groups the uterotubual junction was almost free of granules. This segment, on the contrary, was characterized by the presence of primary and secondary lysosome-like bodies. In the early group the intracellular location of the granules varied between oviduct segments.(ABSTRACT TRUNCATED AT 250 WORDS)

Scanning electron microscopy of in vitro fertilization in cattle.

Cumulus-oocyte complexes were collected from cows at an abattoir by aspiration from small (1-6 mm) follicles. The complexes were matured in vitro for 28 h. Subsequently, the cumulus cells and the zona pellucida were removed by enzyme treatment in a proportion of the complexes (zona-free ova). Both cumulus-enclosed and zona-free ova were inseminated in vitro and processed for scanning electron microscopy after different periods of culture. In the cumulus-enclosed ova the number of spermatozoa attached to and penetrating into the cumulus investment increased with increasing culture time. Practically all spermatozoa displayed intact acrosomes. In the zona-free ova clusters of spermatozoa attached to the ovum surface, and at 5 h a proportion of the spermatozoa had undergone the acrosome reaction, and their internalization into the ooplasma was initiated. The acrosome reaction was characterized by an increasing fenestration of the membrane coverings of the acrosomal region of the sperm head. During the sperm head internalization, where the ovum microvilli appeared to contact especially the equatorial segment and the postacrosomal region, the sperm head gradually disappeared from the ovum surface, and the microvilli at the site of internalization became more bulbous. Simultaneous abstriction of the second polar body was seen in some ova.

Ultrastructural abnormalities of in vitro fertilization of in vitro matured bovine oocytes.

Cumulus-oocyte complexes collected from cows at an abattoir by aspiration from small (1-6 mm) antral follicles were matured and inseminated in vitro. At different time intervals after insemination the ova were processed for transmission electron microscopy. Up to and including 6 h after insemination all ova were unfertilized, and their cortical granules were more or less clustered. At 6 h acrosome reaction of spermatozoa was observed on the surface of the zona pellucida. At 8 h the first fertilized ovum appeared and the first fully developed spherical pronucleus was observed, at 20 h the first apposition of pronuclei was seen, and at 40 h divisions were ongoing or completed. More than one third of the fertilized ova showed polyspermic penetration of the zona pellucida, and in most of these ova different developmental stages of supernumerary pronucleus formation were observed in the ooplasm. Abnormal cortical granule release was seen in approximately half of the fertilized ova, and it was more frequent in ova with polyspermic as opposed to monospermic penetration of the zona pellucida.

Acrosomal status in fresh and frozen/thawed stallion spermatozoa evaluated by scanning electron microscopy.

Semen from stallions with equal fertility at natural services, but yielding semen with either satisfactory or poor fertilizing capability after freezing and thawing, was processed for scanning electron microscopy before and after freezing and thawing. In all fresh semen samples the following three categories of acrosomal defect were noticed: (1) minor fenestrations of the plasma membrane (PM) and outer acrosomal membrane (OAM), (2) complete vesiculation and loss of PM and OAM and (3) lack of a large circular part of PM and OAM. The frequency of these defects ranged from 15% to 27%. All frozen and thawed samples displayed defects of categories 1-3 at similar frequencies as the fresh ones. However, additional defects categorized as: (4) major fenestrations of PM and OAM and (5) complete vesiculation of PM and OAM without loss of the vesicles were noted upon freezing and thawing. The total frequency of defects categorized as 4 and 5 ranged from 8% to 21%, and they seemed to be more frequent in stallions with poor fertility after freezing and thawing although the difference was not significant. Moreover, a particular defect categorized as (6) loosening of the whole acrosomal cap was found exclusively in stallions yielding semen with poor freezability.

Oocyte ultrastructure in bovine primordial to early tertiary follicles.

The aim of the present study was to describe in detail the changes occurring in the cytoplasmic ultrastructure of the bovine oocyte from the onset of growth in the primordial follicle until the completion of growth in the tertiary follicle. Bovine oocytes from primordial, primary, secondary and early to mid-antral follicles were processed and analysed by light and transmission electron microscopy. The primordial follicular oocyte was characterized by numerous coated pits on the oolemma and the accumulation of free and organelle-related smooth (SER) and rough (RER) endoplasmic reticulum, round mitochondria and Golgi complexes around the nucleus, which was located slightly off centre. Up to the secondary follicular stage the oocyte displayed an increase in the number of microvilli, elongated mitochondria and Golgi complexes. During the secondary follicular stage, formation of the zona pellucida, development of gap junctions between the oocyte and the granulosa cells, formation of the cortical granules in the oocyte and reduction in the number of coated pits on the oolemma were seen. In the tertiary follicular oocyte up to 100 microm in diameter, the number of Golgi complexes and lipid droplets increased and the organelles were dislocated to the deep cortical region. During the final growth of the oocyte up to >120 microm, the organelles were dislocated further to the peripheral region, the extent of the free SER and RER compartments were reduced, the number of individual cortical granules increased, hooded mitochondria became abundant and the perivitelline space developed. In conclusion, the growth of the bovine oocyte is associated with the relocation and modulation of a number of cytoplasmic organelles as well as the development of oocyte specific structures such as the zona pellucida and cortical granules.

Ultrastructure of the final nuclear maturation of bovine oocytes in vitro.

Cumulus-oocyte complexes were obtained from cow ovaries by aspiration from small (1-6 mm in diameter) antral follicles after slaughter. Complexes with a compact multilayered cumulus investment were cultured and subsequently processed for electron microscopy after various periods of culture. By morphological criteria the oocytes could be divided into the following sequence of meiotic stages. The oocyte nucleus I stage was characterized by a spherical nucleus located peripherally in the ooplasm while undulation of the nuclear envelope and initial chromatin condensation was seen at the oocyte nucleus II stage. The oocyte nucleus breakdown stage was characterized by formation of long slender projections from the nuclear envelope in which the envelope doubled back on itself, appearance of dense areas and haphazardly oriented microtubules in the nucleus, marked condensation of the chromatin, and dissolution of the nuclear envelope into irregular vesicles and tubules. The condensed chromatin I stage was characterized by the location of condensed chromatin configuration and uniformly oriented microtubules in a dense area peripherally in the ooplasm while the final condensed chromatin II stage was characterized by a gradual invasion of condensed chromatin configurations into a dense area combined with the presence of the first polar body in the perivitelline space.

Are lower fertility bulls necessarily less fertile? Proposals concerning insemination procedures.

This essay argues that current procedures of selection for high fertility bulls may overlook young males of potentially high fertility unless these are tested by modified procedures of insemination. Should this suggestion prove to be true, even if only for a small proportion of young bulls that would not previously have been retained as stud animals, then valuable production genes would be kept in the national herd. Modified procedures of introducing the sperm suspension might include (I) deep intra-uterine insemination, (II) insemination into the functional sperm reservoir in the Fallopian tube isthmus, (III) laparoscopic insemination close to the utero-tubal junction, (IV) intra-peritoneal insemination, (V) insemination under conditions of mild superovulation, and (VI) insemination with smooth muscle stimulants and/or sperm stimulating agents added to the suspension. (VII) The potential value of in vitro fertilization assays, such as the zona-free hamster oocyte sperm incorporation test, is also noted. Even if only one of these approaches were found to be fruitful, its impact could be of major significance for the cattle breeding industry.

Factors affecting survival rates of in vitro produced bovine embryos after vitrification and direct in-straw rehydration.

The aim of this work was to investigate the possibilities of simplification, and to outline the limits of application, of a vitrification method for cow embryos. Morulae and blastocysts were produced by in vitro fertilization of slaughterhouse-derived, in vitro matured oocytes with frozen-thawed bull semen, and subsequent culture on a granulosa cell monolayer. Vitrification was performed by equilibration of embryos with 12.5% ethylene glycol and 12.5% dimethylsulphoxide at 20-22 degrees C for 60 s, then with 25% ethylene glycol and 25% dimethylsulphoxide at 4 degrees C for another 60 s. Embryos were then loaded in straws, placed in liquid nitrogen vapour for 2 min, and then plunged. Straws were thawed in a 22 degrees C water-bath, the embryos were directly rehydrated and further incubated in straw, and were then expelled and cultured in vitro for 72 h. In the first experiment, embryos of different age and developmental stage (Day 5 compacted morulae, Day 6 early blastocysts, Days 6 and 7 blastocysts, Day 7 expanded blastocysts and Day 8 hatched blastocysts) as well as Days 7 and 5 blastocysts previously subjected to partial zone dissection were vitrified. After thawing, the re-expansion rates of blastocysts and zona-dissected embryos did not differ (67 and 87%, respectively), and hatching was more frequent for blastocysts frozen in advanced developmental stages (34, 47 and 63% for early blastocysts, blastocysts and expanded blastocysts, respectively). The re-expansion rate of morulae was lower (10%) and no hatching of these embryos was observed. In the second experiment, Day 7 expanded blastocysts were vitrified using PBS, PBS+albumin, TCM199 and TCM199+calf serum as holding media. No differences in re-expansion and hatching rates were seen. However, when incubation with the concentrated cryoprotectant solution was performed at 20-22 degrees C, the embryo survival rate decreased (PBS+albumin) or no embryo survived (TCM199+calf serum) the vitrification procedure. In the third experiment, Day 7 expanded blastocysts were vitrified, thawed, cultured for 1 day, and then re-expanded embryos were again vitrified and thawed. Out of the 87% that survived the first cycle, 73% re-expanded and 47% hatched following the second vitrification and thawing. These observations prove that the vitrification procedure described is relatively harmless, that it can be used for blastocysts of different developmental stages and that an intact zona is not required to obtain high survival rates.