Successful knock-in of Hypertrophic Cardiomyopathy-mutation R723G into the MYH7 gene mimics HCM pathology in pigs.

Familial Hypertrophic Cardiomyopathy (HCM) is the most common inherited cardiac disease. About 30% of the patients are heterozygous for mutations in the MYH7 gene encoding the ß-myosin heavy chain (MyHC). Hallmarks of HCM are cardiomyocyte disarray and hypertrophy of the left ventricle, the symptoms range from slight arrhythmias to sudden cardiac death or heart failure. To gain insight into the underlying mechanisms of the diseases' etiology we aimed to generate genome edited pigs with an HCM-mutation. We used TALEN-mediated genome editing and successfully introduced the HCM-point mutation R723G into the MYH7 gene of porcine fibroblasts and subsequently cloned pigs that were heterozygous for the HCM-mutation R723G. No off-target effects were determined in the R723G-pigs. Surprisingly, the animals died within 24 h post partem, probably due to heart failure as indicated by a shift in the a/ß-MyHC ratio in the left ventricle. Most interestingly, the neonatal pigs displayed features of HCM, including mild myocyte disarray, malformed nuclei, and MYH7-overexpression. The finding of HCM-specific pathology in neonatal R723G-piglets suggests a very early onset of the disease and highlights the importance of novel large animal models for studying causative mechanisms and long-term progression of human cardiac diseases.

Gene-specific profiling of DNA methylation and mRNA expression in bovine oocytes derived from follicles of different size categories.

Epigenetic changes, such as DNA methylation, play an essential role in the acquisition of full developmental competence by mammalian oocytes during the late follicular growth phase. Here we used the bovine model to investigate the DNA methylation profiles of seven candidate genes (imprinted: bH19, bSNRPN; non-imprinted: bZAR1, bDNMT3A, bOCT4, bDNMT3 Lo and bDNMT3 Ls) and the mRNA expression of nine candidate genes (imprinted: bSNRPN, bPEG3, bIGF2R; non-imprinted: bPRDX1, bDNMT1B, bDNMT3A, bZAR1, bHSF1 and bNLRP9) in oocytes from antral follicles of three different size classes (≤2mm, 3-5mm, ≥6mm) to unravel the epigenetic contribution to this process. We observed an increased number of aberrantly methylated alleles in bH19, bSNRPN and bDNMT3 Lo of oocytes from small antral follicles (≤2mm), correlating with lower developmental competence. Furthermore, we detected an increased frequency of CpG sites with an unclear methylation status for DNMT3 Ls, specifically in oocytes from follicles ≥6mm, predominantly at three CpG positions (CpG2, CpG7 and CpG8), of which CpG7 is a potential regulatory site. No major differences in mRNA expression were observed, indicating that the transcriptional machinery may not yet be active during the follicular growth phase. Our results support the notion that a follicle diameter of ~2mm is a critical stage for establishing DNA methylation profiles and indicate a link between DNA methylation and the acquisition of oocyte developmental competence.

An air-liquid interphase approach for modeling the early embryo-maternal contact zone.

We developed an air-liquid interphase culture procedure for mammalian oviduct epithelial cells leading to the formation of functional epithelial tissues, which generate oviduct fluid surrogates. These in vitro oviduct epithelia can be co-cultured with living zygotes and enable embryonic development up to the blastocyst stage without addition of embryo culture medium. The described strategy is broadly applicable to analyze early embryo-maternal interactions under standardized in vitro conditions.

Satellite DNA methylation status and expression of selected genes in Bos indicus blastocysts produced in vivo and in vitro.

Bovine embryos produced in vivo and in vitro differ with respect to molecular profiles, including epigenetic marks and gene expression profiles. This study investigated the CpG methylation status in bovine testis satellite I (BTS) and Bos taurus alpha satellite I (BTαS) DNA sequences, and concomitantly the relative abundance of transcripts, critically involved in DNA methylation (DNMT1 and DNMT3A), growth and development (IGF2R) and pluripotency (POU5F1) in Bos indicus embryos produced in vitro or in vivo. Results revealed that methylation of BTS were higher (P < 0.05) in embryos produced in vitro compared with their in vivo produced counterparts, while the methylation status of BTαS was similar in both groups. There were no significant differences in transcript abundance for DNMT3A, IGF2R and POU5F1 between blastocysts produced in vivo and in vitro. However, a significantly lower amount of DNMT1 transcripts was found in the in vitro cultured embryos (P < 0.05) compared with their in vivo derived counterparts. In conclusion, this study reported only minor changes in the expression of developmentally important genes and satellite DNA methylation related to the in vitro embryo production system.

siRNA mediated knockdown of tissue factor expression in pigs for xenotransplantation.

Acute vascular rejection (AVR), in particular microvascular thrombosis, is an important barrier to successful pig-to-primate xenotransplantation. Here, we report the generation of pigs with decreased tissue factor (TF) levels induced by small interfering (si)RNA-mediated gene silencing. Porcine fibroblasts were transfected with TF-targeting small hairpin (sh)RNA and used for somatic cell nuclear transfer. Offspring were analyzed for siRNA, TF mRNA and TF protein level. Functionality of TF downregulation was investigated by a whole blood clotting test and a flow chamber assay. TF siRNA was expressed in all twelve liveborn piglets. TF mRNA expression was reduced by 94.1 ± 4.7% in TF knockdown (TFkd) fibroblasts compared to wild-type (WT). TF protein expression in PAEC stimulated with 50 ng/mL TNF-α was significantly lower in TFkd pigs (mean fluorescence intensity TFkd: 7136 ± 136 vs. WT: 13 038 ± 1672). TF downregulation significantly increased clotting time (TFkd: 73.3 ± 8.8 min, WT: 45.8 ± 7.7 min, p < 0.0001) and significantly decreased thrombus formation compared to WT (mean thrombus coverage per viewing field in %; WT: 23.5 ± 13.0, TFkd: 2.6 ± 3.7, p < 0.0001). Our data show that a functional knockdown of TF is compatible with normal development and survival of pigs. TF knockdown could be a valuable component in the generation of multi-transgenic pigs for xenotransplantation.

Somatic cell nuclear transfer cloning: practical applications and current legislation.

Somatic cloning is emerging as a new biotechnology by which the opportunities arising from the advances in molecular genetics and genome analysis can be implemented in animal breeding. Significant improvements have been made in SCNT protocols in the past years which now allow to embarking on practical applications. The main areas of application of SCNT are: Reproductive cloning, therapeutic cloning and basic research. A great application potential of SCNT based cloning is the production of genetically modified (transgenic) animals. Somatic cell nuclear transfer based transgenic animal production has significant advances over the previously employed microinjection of foreign DNA into pronuclei of zygotes. This cell based transgenesis is compatible with gene targeting and allows both, the addition of a specific gene and the deletion of an endogenous gene. Efficient transgenic animal production provides numerous opportunities for agriculture and biomedicine. Regulatory agencies around the world have agreed that food derived from cloned animals and their offspring is safe and there is no scientific basis for questioning this. Commercial application of somatic cloning within the EU is via the Novel Food regulation EC No. 258/97. Somatic cloning raises novel questions regarding the ethical and moral status of animals and their welfare which has prompted a controversial discussion in Europe which has not yet been resolved.

The role of RNA polymerase I transcription and embryonic genome activation in nucleolar development in bovine preimplantation embryos.

The aim of the present study was to investigate the role of RNA polymerase I (RPI) transcription in nucleolar development during major transcriptional activation (MTA) in cattle. Late eight-cell embryos were cultured in the absence (control group) or presence of actinomycin D (AD) (RPI inhibition, AD 0.2 microg/ml; total transcriptional inhibition, AD 2.0 microg/ml). Late four-cell embryos were cultured to late eight-cell stage in 0.2 microg/ml AD (MTA prevention, ADLT (long-term total transcriptional inhibition group). Embryos were processed for autoradiography, transmission electron microscopy, fluorescent in situ hybridization (ribosomal RNA, rRNA), silver staining (nucleolar proteins), and immunofluorescence (RPI). Control embryos displayed extranucleolar and nucleolar transcription, functional nucleoli, and distinct RPI localization. Nuclei (97%) showed large rRNA clusters, in 94.1% co-localized with nucleolar proteins deposits. In AD 0.2 group, only extranucleolar transcription was detected. Segregated dense-fibrillar and granular components, but no fibrillar centers, were observed. RPI was dispersed. Nuclei (55%) presented rRNA clusters, in 38.8% co-localized with silver-stained deposits. AD 2.0 and ADLT groups displayed no transcription and disintegrating nucleolar precursors. AD 2.0 (34%) and 14% (ADLT) of nuclei presented clusters of maternally inherited rRNA. In AD 2.0 group, RPI was dispersed, but 17.2% of nuclei showed colocalization of rRNA with nucleolar proteins. In ADLT group, RPI was lacking and clustering of nucleolar proteins was hampered. In conclusion, rDNA transcription is not required for targeting of rRNA processing proteins, rRNA is maternally inherited and target to rDNA independent of transcription, and de novo transcription is required for proper nucleologenesis in cattle.

Altered mRNA expression patterns in bovine blastocysts after fertilisation in vitro using flow-cytometrically sex-sorted sperm.

Sperm-sexing has been used to produce embryos and offspring of a pre-determined sex in a number of species. However, the fertility of sex-sorted sperm is reduced and the full effects of sperm-sexing remain to be elucidated. The purpose of the present study was to investigate the potential effects of sex-sorted sperm on mRNA expression patterns of developmentally important genes employing in vitro produced bovine embryos. Bovine embryos were produced in vitro with unsorted and sex-sorted sperm and mRNA expression patterns were determined for glucose-3 transporter (Glut-3), glucose-6-phosphate dehydrogenase (G6PD), X-inactive specific transcript (X-ist) and Heat shock protein 70.1 (Hsp) using semi-quantitative endpoint reverse transcriptase-PCR in male and female, day-7 and 8 embryos. The relative abundance (RA) of Glut-3 was higher for day-7 male than female embryos, and day-7 embryos derived from unsorted compared with sex-sorted sperm. The RA of G6PD was higher for embryos derived from unsorted than sex-sorted sperm, and for day-8 female compared with male embryos. The RA of Xist was higher for female than male embryos, and for day-7 female embryos derived from unsorted than sex-sorted sperm. Hsp RA was higher for female compared with male embryos, was similar for day-7 and 8 embryos, and unsorted and sex-sorted sperm derived embryos. These results demonstrate differential expression of developmentally important genes between male and female embryos, and embryos derived from unsorted and sex-sorted sperm.

Isolation of bovine cardiomyocytes for reprogramming studies based on nuclear transfer.

The goal of this study was to establish and validate a protocol for preparing bovine cardiomyocytes from slaughterhouse material for nuclear transfer experiments. The cardiomyocyte was selected because it is a terminally differentiated cell and strongly expresses a unique subset of genes which can be monitored during the reprogramming period. A total of 39 trials were conducted, and an optimized protocol was developed yielding individual contractile cardiomyocytes from 3-5-month-old bovine fetuses The basic protocol involves stabilization of bovine heart tissue for transportation from the slaughterhouse to the laboratory by perfusion with Custodiol. This was followed by an enzymatic dissociation with collagenase in calcium-free medium and yielded individual contractile rod-shaped cardiomyocytes. Subsequent addition of Ca2+ caused the cardiomyocytes to round up which was an essential pre-condition for drawing them into glass transfer pipettes for delivery into the perivitelline space and for efficient electrofusion with cytoplasts derived from in vitro matured bovine oocytes. The use of cardiomyocytes maintained at 37 degrees C in nuclear transfer, resulted in a significantly reduced proportion of blastocysts compared to adult fibroblasts (14.0% versus 32.7%). Storage of cardiomyocytes at 4 degrees C prior to nuclear transfer was not compatible with blastocyst development. It is expected that this system will be valuable for investigating the reprogramming of gene expression which occurs after somatic cell nuclear transfer.

Gene expression patterns in in vitro-produced and somatic nuclear transfer-derived preimplantation bovine embryos: relationship to the large offspring syndrome?

A considerable proportion of the offspring born from somatic nuclear transfer (sNT)-derived and in vitro-produced (IVP) embryos, particularly in ruminants and mice, is affected by multiple abnormalities of which a high birth weight is the predominant feature; a phenomenon that has been called "large offspring syndrome (LOS)". The underlying mechanisms are largely unknown at present, but changes in epigenetic modifications occurring during preimplantation development resulting in perturbed embryonic and fetal gene expression patterns are thought to be involved in the syndrome. This review summarizes results from studies comparing mRNA expression patterns from IVP and sNT-derived embryos to those of their in vivo counterparts, which are regarded as the "gold standard". Numerous aberrations have been observed ranging from suppression of expression to de novo overexpression or more frequently to a significant up- or down-regulation of a specific gene. These observations emphasize the need for further studies during preimplantation embryo development to gain insight in the molecular, preferentially epigenetic, mechanisms regulating embryonic and fetal development. Understanding these mechanisms will help to improve biotechnologies applied to early embryos in all species including humans.

Gene expression patterns in bovine in vitro-produced and nuclear transfer-derived embryos and their implications for early development.

Bovine in vitro-produced (IVP) and nuclear transfer (NT)-derived embryos differ from their in vivo-developed counterparts in a number of characteristics. A preeminent observation is the occurrence of the large offspring syndrome, which is correlated with considerable embryonic fetal and postnatal losses. We summarize here results from our studies in which we compared gene expression patterns from IVP and NT-derived embryos with those from their IVP counterparts. Numerous aberrations were found in IVP and NT-derived embryos, including a complete lack of expression, an induced expression, or a significant up- or downregulation of a specific gene. These alterations may affect a number of physiological functions and are considered as a kind of stress response of the embryos to deficient environmental conditions. We hypothesize that the alterations are caused by epigenetic modifications, primarily by changes in the methylation patterns. Unravelling these epigenetic modifications is promising to reveal the underlying mechanisms of the large offspring syndrome.

In vitro production and nuclear transfer affect dosage compensation of the X-linked gene transcripts G6PD, PGK, and Xist in preimplantation bovine embryos.

Equal expression of X-linked genes such as G6PD and PGK in females and males and the initiation of X-chromosome inactivation are critically dependent on the expression of the X-inactive specific transcript (Xist). The objective of the present study was to determine the effects of in vitro production (IVP) and nuclear transfer (NT) on the relative abundance (RA) of the X-linked transcripts G6PD, PGK, and Xist in preimplantation bovine embryos. In experiment 1, sex-determined IVP or in vivo-produced embryos were analyzed for mRNA expression of the 3 genes. The sex ratio was 36% vs. 64% in IVP blastocysts and thus deviated significantly from the expected ratio of 50% in the vivo control group. The RA of G6PD transcripts was significantly higher in female IVP embryos than in male embryos. In contrast, no significant differences were seen between in vivo-derived female embryos and their male counterparts. At the morula stage, female IVP embryos transcribed significantly more PGK mRNA than did male embryos. However, blastocysts did not exhibit significant differences in PGK transcripts. No differences were observed for in vivo-derived embryos with regard to the RA of PGK transcripts. The RA of Xist mRNA was significantly higher in all female embryos than in their male counterparts. In experiment 2, IVP, in vivo-developed, NT-derived, and parthenogenetic embryos carrying two X chromosomes of either maternal and paternal origin or of maternal origin only (parthenogenotes) were analyzed for the RA of the 3 genes. In NT-derived morulae, the RA of G6PD transcripts was significantly increased compared with their IVP and in vivo-generated counterparts. G6PD transcript levels were significantly increased in IVP blastocysts compared with in vivo-generated and parthenogenetic embryos. At the morula stage, PGK transcripts were similar in all groups, but the RA of PGK transcripts was significantly higher in IVP blastocysts than in their in vivo-generated, parthenogenetic, and NT-derived counterparts. The RA of Xist was significantly elevated in NT-derived morulae compared with IVP, in vivo-generated, and parthenogenetic embryos. NT-derived blastocysts showed an increased Xist expression compared with that of IVP, in vivo-generated, and parthenogenetic embryos. Results of the present study show for the first time that differences in X-chromosome-linked gene transcript levels are related to a perturbed dosage compensation in female and male IVP and female NT-derived embryos. This finding warrants further studies to improve IVP systems and NT protocols to ensure the production of embryos with normal gene expression patterns.

A detailed analysis of pronucleus development in bovine zygotes in vitro: cell-cycle chronology and ultrastructure.

The aim of the present experiment was to analyze the chronology of pronucleus development and DNA synthesis, as well as the ultrastructure of intranuclear bodies, in bovine zygotes produced in vitro. Bovine oocytes were matured and fertilized in vitro, and sperm penetration and pronucleus development were examined. DNA synthesis was investigated by sequential incubation with [3H]- and [14C]thymidine followed by autoradiography on semithin sections. Ultrathin sections for transmission electron microscopy were prepared from the same zygotes. Sperm penetration was noted for the first time at 4 hr after in vitro insemination and reached a maximum at 6 hr. Pronucleus formation was initiated at 4 hr, and up to at least 11 hr the maternal pronucleus was more developed than its paternal counterpart. DNA synthesis was initiated at 14-15 hr, and the S-phase lasted for 8-10 hr. The most prominent ultrastructural entities of the pronuclei were the nucleolus precursor bodies (NPBs). During the S- and G2-phases, the NPBs spatially associated with clusters of interchromatin-like granules. The two components were firmly attached to each other by an electron-dense reticulum. During the late G2-phase, the NPBs were apparently detached from the interchromatin-like granules and the electron-dense reticulum again. The interaction between the intranuclear bodies and granules appears to be comparable with the situation previously described for in vivo-produced bovine zygotes (J Laurincík et al., Mol Reprod Dev 43:62-69, 1996), except for the lack of vacuolization of the NPBs during the S-phase in vitro.

[Status of development and areas of employment of in vitro production of cattle embryos]. / Entwicklungsstand und Anwendungsbereiche der In vitro Produktion von Rinderembryonen.

Efficient methods for the collection of oocytes derived from slaughterhouse ovaries and living animals are available. Maturation, fertilization and culture of bovine oocytes/embryos can be performed in vitro with high success rates. The in vitro culture from the fertilized oocyte up to the blastocyst stage currently is the most limiting step of the whole procedure. The establishment of a completely defined culture system would allow the identification of the physiological requirements for preimplantation embryo development and the stepwise improvement of the in vitro production system. The physiological integrity of in vitro produced embryos compared to their in vivo counterparts can be assessed either by molecular biological techniques or in metabolism studies. The in vitro production of bovine embryos with its variety of areas of applications is a useful tool in maintenance and improvement of competitiveness of future cattle breeding.

Collection of oocytes from cattle via follicular aspiration aided by ultrasound with or without gonadotropin pretreatment and in different reproductive stages.

Ultrasound-guided follicular aspiration was performed on 29 Holstein-Friesian cows/heifers twice weekly at 3- to 4-d intervals over a period of 2 consecutive estrous cycles (total 42 d). For visualization of the ovaries and guidance of the aspiration needle, a 6.5 MHz fingertip probe on a 62 cm probe carrier was inserted into the vagina. The disposable aspiration needle was connected to a permanent rinse tubing system, thus ensuring minimum death of oocytes in the aspiration processs. After penetration of the vaginal wall, the needle was inserted into a follicle of the rectally fixed ovary. Cumulus oocyte complexes (COC) were aspirated at a pressure of 100 mm Hg. In the first experiment, the effect of an additional gonadotropin treatment 4 d prior to aspiration was investigated in 8 lactating cows. Following FSH-treatment, the number of aspirated follicles was higher (P < 0.05) than in the nontreated animals (10.6 +/- 0.7 vs 8.9 +/- 0.5). The number of recovered COC (7.0 +/- 0.6 vs 5.8 +/- 0.5), the recovery rate (COC per aspirated follicle) (66.6% vs 65.4%), the percentage of viable COC (56.8% vs 52.1%), the cleavage rate upon in vitro maturation and in vitro fertilization (56.7% vs 59.8%) as well as the rate of morula/blastocyst formation (3.8% vs 2.9%) were similar in both groups. In the second experiment, follicles were aspirated in 4 lactating cows, 6 dry cows, 4 pregnant cows (first 35 d of pregnancy), and 4 heifers. The average number of aspirated follicles and recovered COC was higher (P < 0.05) in the first 2 groups (10.6 +/- 0.6 and 9.3 +/- 0.7 follicles; 7.2 +/- 0.5 and 6.9 +/- 0.7 oocytes) than in trie 2 other treatment groups (7.3 +/- 0.5 and 8.1 +/- 0.5 follicles; 5.0 +/- 0.4 and 5.7 +/- 0.5 oocytes). The percentage of viable COC was higher (P < 0.05; 68.3%) in lactating animals than in all the other groups (49.7, 52.5 and 57.4%, respectively). Similarly, upon in vitro fertilization, cleavage rate was higher (P < 0.05; 63.4%) in lactating cows than in the other groups (43.7, 50.5, 55.1%, respectively). A total of 21.5, 22.7, 11.9 and 13.5%, respectively, in the 4 groups of the in vitro fertilized oocytes reached the morula and blastocyst stages. After transfer of a total of 48 embryos 22 pregnancies (45.8%) were established as detected on Day 65. We conclude that 1) repeated aspiration of viable COC at short intervals is possible, 2) additional FSH-treatment does not increase oocyte yields, and 3) viable blastocysts can be produced from cattle at various reproductive phases irrespective of the reproductive phase.

Transcriptional activity and nuclear ultrastructure of 8-cell bovine embryos developed by in vitro maturation and fertilization of oocytes from different growth categories of antral follicles.

The normalcy of nuclear differentiation in 8-cell bovine embryos derived by in vitro procedures from oocytes isolated from antral follicles of three different size categories was studied using autoradiographic localization of RNA synthesis and fine-structure nuclear morphology. In the few embryos derived from the oocytes from the small category of follicles (1-2 mm) the unlabeled nuclei prevailed. In contrast, the oocytes isolated from the more progressed follicles (medium and large size: 2-8 mm) yielded embryos with only slight, if at all detectable, differences to the picture expected in in vivo developed normal embryos. The diverging features probably concerned a slower onset of gene transcription, its consecutive pattern of localization as well as less pronounced progress of chromatin condensation and nucleolar differentiation. The assessment of these morphological changes and characteristics for individual embryos was obscured by marked differences in the status of the nuclei of particular blastomeres forming an embryo. Here the differences were seen in the fragmentation of nuclei, in the degree of chromatin condensation and in the absence of the nucleolus-associated chromatin in some of the blastomeres. It is suggested that most of the embryos from medium and large follicles have a comparable differentiation pattern of nuclear function development as embryos developing normally in vivo.

Fertilization and developmental competence of bovine oocytes derived from different categories of antral follicles.

This study investigated the capacity of healthy oocytes derived from follicles of different size to undergo normal fertilization and early embryonic development in vitro and full-term development in vivo. Ovaries were collected from a local abattoir and dissected and classified as follows: group A, greater than 4-8 mm (large); group B, greater than 2-4 mm (medium); and group C, greater than 1-2 mm (small). Oocytes were isolated by puncturing the follicular wall and pressing of the follicle. Only healthy-looking cumulus-oocyte complexes (COC) were used for in vitro maturation. Oocytes were fertilized in vitro by frozen/thawed semen from one bull. Approximately one-fourth of all oocytes was fixed and stained 15-20 h after fertilization, to determine penetration rates. The remaining eggs were transferred to culture medium and were cultivated for up to 9.5 days. Cleavage was observed 65 h and 7 days after fertilization. Expanded, hatching, and hatched blastocysts were fixed and stained after 9.5 days of culture. A total of 86 blastocysts derived from group A and B oocytes was nonsurgically transferred to synchronized recipients 7-8 days after onset of culture. A total of 6.624 follicles were dissected from 265 ovaries, and 1,485 oocytes were isolated from 1,671 group A follicles, 3,509 oocytes from 3,862 group B follicles, and 965 oocytes from 1,091 group C follicles. The fertilization rate, rate of normal fertilization, rate of polyspermy, and rate of other abnormal fertilization features were as follows: group A, 84.9%, 43.2%, 34.1%, 7.6%; group B, 83.6%, 44.8%, 31.1%, 7.8%; and group C, 61.7%, 13.1%, 33.7%, 19.1%.(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro survival of fresh and frozen/thawed bovine demi-embryos.

Three experiments were conducted to investigate the effects of type of culture medium in freshly bisected bovine embryos and the effects of agar embedding and of 1.2 propanediol (PROH) as the cryoprotectant in frozen/thawed bisected bovine embryos on development in vitro. A total of 265 bovine embryos were used as controls or were microsurgically bisected and were cultured in vitro for 48 hours and development was determined 24 and 48 hours after the onset of culture. Whitten's medium supported more (P<0.05) intact and demi-embryos to grow to expanded blastocysts (92.9 and 73.1%, respectively) compared with Ham's F10 (43.8 and 26.3%, respectively) and PBS (53.8 and 12.5%, respectively). Embedding in agar and culture in Whitten's medium resulted in a higher (P<0.05) percentage of in vitro development of frozen/thawed demi-embryos after 24 hours than the freezing of nonembedded demi-embryos (44.1 versus 19.6%, respectively). This difference disappeared, however, after a 48 hours culture period (17.6 versus 11.8%, respectively). Following freezing in PROH, survival rates of 40 and 28%, respectively after 24 hours of culture were obtained for intact and demi-embryos. The respective percentages after 48 hours were 8.6 and 16%. Since neither embedding in agar nor the use of PROH as the cryoprotectant resulted in high survival rates of frozen/thawed demi-embryos in vitro, new freezing procedures are needed to overcome the sensitivity of demi-embryos to freezing and thawing.