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Pigs share anatomical and physiological traits with humans and can serve as a large-animal model for translational medicine. Bona fide porcine pluripotent stem cells (PSCs) could facilitate testing cell and drug therapies. Agriculture and biotechnology may benefit from the ability to produce immune cells for studying animal infectious diseases and to readily edit the porcine genome in stem cells. Isolating porcine PSCs from preimplantation embryos has been intensively attempted over the past decades. We previously reported the derivation of expanded potential stem cells (EPSCs) from preimplantation embryos and by reprogramming somatic cells of multiple mammalian species, including pigs. Porcine EPSCs (pEPSCs) self-renew indefinitely, differentiate into embryonic and extra-embryonic lineages, and permit precision genome editing. Here we present a highly reproducible experimental procedure and data of an optimized and robust porcine EPSC culture system and its use in deriving new pEPSC lines from preimplantation embryos and reprogrammed somatic cells. No particular expertise is required for the protocols, which take ~4-6 weeks to complete. Importantly, we successfully established pEPSC lines from both in vitro fertilized and somatic cell nuclear transfer-derived embryos. These new pEPSC lines proliferated robustly over long-term passaging and were amenable to both simple indels and precision genome editing, with up to 100% targeting efficiency. The pEPSCs differentiated into embryonic cell lineages in vitro and teratomas in vivo, and into porcine trophoblast stem cells in human trophoblast stem cell medium. We show here that pEPSCs have unique epigenetic features, particularly H3K27me3 levels substantially lower than fibroblasts., (© 2024. Springer Nature Limited.)

Cyclic adenosine monophosphate (cAMP) modulators have been used to avoid spontaneous oocyte maturation and concomitantly improve oocyte developmental competence. The current work evaluated the effects of the addition of cAMP modulators forskolin, 3-isobutyl-1-methylxanthine (IBMX) and cilostamide during in vitro maturation on the quality and yields of blastocysts. The following experimental groups were evaluated: (i) slicing or (ii) aspiration and maturation in tissue culture medium (TCM)199 for 24 h (TCM24slicing and TCM24aspiration, respectively), (iii) aspiration and maturation in the presence of cAMP modulators for 30 h (cAMP30aspiration) and in vivo-produced blastocysts. In vitro-matured oocytes were fertilized and presumptive zygotes were cultured in vitro to assess embryo development. Cleavage, blastocyst formation, blastocyst cell number, mRNA abundance of selected genes and global methylation profiles were evaluated. Blastocyst rate/zygotes for the TCM24aspiration protocol was improved (32.2 ± 2.1%) compared with TCM24slicing and cAMP30aspiration (23.4 ± 1.2% and 23.3 ± 2.0%, respectively, P 0.05), while those from the other groups were significantly elevated. It is concluded that retrieval, collection systems and addition of cAMP modulators can affect oocyte developmental competence, which is reflected not only in blastocyst rates but also in global DNA methylation and gene expression patterns.

Lipids play fundamental roles in mammalian embryo preimplantation development and cell fate. Triacylglycerol accumulates in oocytes and blastomeres as lipid droplets, phospholipids influence membrane functional properties, and essential fatty acid metabolism is important for maintaining the stemness of cells cultured in vitro. The growing impact that lipids have in the field of developmental biology makes analytical approaches to analyse structural information of great interest. This paper describes the concept and presents the results of lipid profiling by mass spectrometry (MS) of oocytes and preimplantation embryos, with special focus on ambient ionisation. Based on our previous experience with oocytes and embryos, we aim to convey that ambient MS is also valuable for stem cell differentiation analysis. Ambient ionisation MS allows the detection of a wide range of lipid classes (e.g. free fatty acids, cholesterol esters, phospholipids) in single oocytes, embryos and cell pellets, which are informative of in vitro culture impact, developmental and differentiation stages. Background on MS principles, the importance of underused MS scan modes for structural analysis of lipids, and statistical approaches used for data analysis are covered. We envisage that MS alone or in combination with other techniques will have a profound impact on the understanding of lipid metabolism, particularly in early embryo development and cell differentiation research.

Alteration of maternal lipid metabolism early in development has been shown to trigger obesity, insulin resistance, type 2 diabetes and cardiovascular diseases later in life in humans and animal models. Here, we set out to determine (i) lipid composition dynamics in single oocytes and preimplantation embryos by high mass resolution desorption electrospray ionization mass spectrometry (DESI-MS), using the bovine species as biological model, (ii) the metabolically most relevant lipid compounds by multivariate data analysis and (iii) lipid upstream metabolism by quantitative real-time PCR (qRT-PCR) analysis of several target genes (ACAT1, CPT 1b, FASN, SREBP1 and SCAP). Bovine oocytes and blastocysts were individually analyzed by DESI-MS in both positive and negative ion modes, without lipid extraction and under ambient conditions, and were profiled for free fatty acids (FFA), phospholipids (PL), cholesterol-related molecules, and triacylglycerols (TAG). Principal component analysis (PCA) and linear discriminant analysis (LDA), performed for the first time on DESI-MS fused data, allowed unequivocal discrimination between oocytes and blastocysts based on specific lipid profiles. This analytical approach resulted in broad and detailed lipid annotation of single oocytes and blastocysts. Results of DESI-MS and transcript regulation analysis demonstrate that blastocysts produced in vitro and their in vivo counterparts differed significantly in the homeostasis of cholesterol and FFA metabolism. These results should assist in the production of viable and healthy embryos by elucidating in vivo embryonic lipid metabolism.

Normal development depends on the precise sequence of changes in the configuration of chromatin; these are primarily related to specific biochemical modifications such as acetylation or methylation of histones and DNA methylation. While the role of DNA methylation during preimplantation development has been studied extensively, little is known about histone modifications related to early embryonic development. Here, we investigated gene-specific histone modifications in in vitro produced bovine blastocysts. Selected genes thought to be critical for bovine preimplantation development were examined and included POU5F1 (OCT4), NANOG, INFT, GAPDH, SLC2A3 and IGF1. We used chromatin immunoprecipitation from pools of bovine blastocysts to unravel several modifications of histone H3 in relation to mRNA expression profiles. We focused on the two cell compartments of the blastocyst, the inner cell mass (ICM) and the trophectoderm (TE). We show that gene expression patterns in the ICM and TE of the bovine blastocyst are consistent with histone modification patterns on the promoter of the corresponding genes. The data show a complex epigenetic pattern of promoter occupancy by transcriptionally permissive and repressive H3 modifications. These results pave the way to in-depth epigenetic studies of preimplantation embryos that are crucial to gain a better understanding of the epigenetic changes frequently observed after use of assisted reproductive technologies.

Telomeres play an important role in aging, and are critical for the regenerative capacity of mammalian cells. The holoenzyme telomerase rebuilds telomeres and is composed of two components, the catalytic protein telomerase reverse transcriptase (TERT) and the telomerase RNA (TERC). TERC is ubiquitously expressed in somatic cells and is thought to have no regulatory effects on telomerase activity. Transgenic expression of human TERT (hTERT) in bovine somatic and embryonic cells extends telomere length and enhances telomerase activity. To obtain further insight into the regulatory capacity of the two telomerase components, we have studied the ability of hTERC and hTERT to increase telomerase activity and telomere length in bovine embryos. Expression plasmids for the human RNA component (hTERC) and/or the catalytic subunit of human telomerase (hTERT), respectively, were injected into the cytoplasm of in vitro-produced bovine zygotes. Ectopic expression of hTERC increased telomerase activity and telomere length in bovine blastocysts. Coexpression of hTERT and hTERC did not result in further telomere elongation when compared to the hTERC group. These data indicate that TERC is one of the limiting factors of telomerase activity in bovine blastocysts, and further establish bovine preimplantation embryos as a useful model to modulate telomere length with impact for basic embryology and derivation of pluripotent cells.

The enzyme telomerase is active in germ cells and is critically involved in maintenance of telomere length in successive generations. In preimplantation mammalian embryos, telomerase activity is present from the morula stage onward and is associated with an increase in telomere length in blastocysts. Herein, we show that telomere length regulation in murine and bovine blastocysts differed between trophectodermal and inner cell mass cells in a species-specific manner. Ectopic expression of human telomerase reverse transcriptase (TERT) in bovine embryos increased telomerase activity and in turn increased telomere length. Transient expression of human TERT could be targeted to the 4-cell to morula stages and to the morula to blastocyst stages using unmodified and cytosine-methylated expression plasmids, respectively. Introduction of human TERT constructs in bovine embryos resulted in functional telomerase expression and effective telomere elongation, allowing us to study the effects on embryonic development. Ultimately, these studies may lead to a large-animal model for telomere regulation and aging.

DNA demethylation and remethylation are crucial for reprogramming of the differentiated parental/somatic genome in the recipient ooplasm upon somatic cell nuclear transfer. Here, we analyzed the DNA methylation dynamics during porcine preimplantation development. Porcine in vivo developed (IV), in vitro fertilized (IVF), somatic cell nuclear transfer (SCNT) and parthenogenetically activated (PA) embryos were evaluated for DNA methylation quantification at different developmental stages. Fertilized (IV and IVF) one-cell stages lacked a substantial active demethylation of the paternal genome. Embryos produced under in vitro conditions had higher levels of DNA methylation than IV. A lineage-specific DNA methylation (hypermethylation of the inner cell mass and hypomethylation of the trophectoderm) was observed in porcine IV late blastocysts, but was absent in PA- and SCNT-derived blastocysts despite the occurrence of de novo methylation in early blastocysts. Comparable levels of DNA methylation were found in IV embryos and in 50% and 14% of SCNT early and late blastocysts, respectively. In conclusion, DNA methylation patterns were adversely affected by in vitro embryo production.

The recent identification of an intragenic differentially methylated region (DMR) within the last exon of the bovine Insulin-like growth factor 2 (IGF2) gene provides a diagnostic tool for in-depth investigation of bovine imprinting and regulatory mechanisms which are active during embryo development. Here, we used bisulfite sequencing to compare sex-specific DNA methylation patterns within this DMR in bovine blastocysts produced in vivo, by in vitro fertilization and culture, SCNT, androgenesis or parthenogenesis. In in vivo derived embryos, DNA methylation was removed from this intragenic DMR after fertilization, but partially replaced by the time the embryo reached the blastocyst stage. Among embryos developing in vivo, the level of DNA methylation was significantly lower in female than in male blastocysts. This sexual dimorphism was also found between parthenogenetic and androgenetic embryos, and followed the donor cell sex in SCNT derived blastocysts and is evidence for correct methylation reprogramming in SCNT embryos.

Histone modification genes in bovine embryos: The mRNA expression pattern of histone-related genes was determined in bovine oocytes and embryos. We compared immature and in vitro-matured oocytes, either before or after enucleation and activation, in vitro produced embryos (zygotes, 8-16 cell stages, blastocysts), embryos cloned with female or male donor cells; parthenogenetic embryos, and in vivo-derived blastocysts to detect deviations from the normal expression pattern. A sensitive semi-quantitative endpoint RT-PCR assay was used to reveal differences in histone deacetylation [histone deacetylase 2 (HDAC2)]; histone acetylation [histone acetyltransferase 1 (HAT1)]; histone methylation [histone methyltransferases (SUV39H1, G9A)]; heterochromatin formation [heterochromatin protein 1 (HP1)]; and chromatin-mediated transcription regulation [zygote arrest 1 (ZAR1)]. With the exception of ZAR1, these mRNAs were present throughout preimplantation development. The relative abundance of mRNAs for histone methyltransferases (SUV39H1 and G9A) and for heterochromatin-associated protein (HP1) differed significantly before and after activation of the bovine embryonic genome. The similarity of HAT1 gene expression in 8-16 cell embryos and blastocysts suggests that histone acetylation is primarily affected by in vitro culture only prior to embryonic genome activation. HDAC2 gene mRNA expression was not affected by in vitro culture and/or cloning before and after activation of the embryonic genome. The donor cell line affected mRNA expression patterns of genes involved in reprogramming cloned embryos suggesting epigenetic dysregulation. Results show that both in vitro production and somatic cloning alter the mRNA expression of histone modifying genes in bovine embryos., ((c) 2007 Wiley-Liss, Inc.)

Addition of insulin-like growth factor-1 (IGF-1) to culture medium increases the proportion of bovine embryos that develop to the blastocyst stage and increases embryo survival following transfer to heat-stressed, lactating dairy cows. The objective of the present study was to determine molecular and cellular correlates of these actions of IGF-1. Embryos were produced in vitro and cultured for 7 days with or without 100 ng/ml IGF-1. On d 7 after insemination, grade 1 expanded blastocysts were harvested and used to determine total cell number, percent apoptosis, cell allocation to the inner cell mass and trophectoderm, and the relative abundance of several developmentally important gene transcripts. There was no significant effect of IGF-1 treatment on blastocyst cell number, the proportion of blastomeres that were apoptotic, or the number of cells in the inner cell mass and trophectoderm. However, differences in the relative abundance of several mRNA transcripts were observed between control and IGF-1 treated embryos. Addition of IGF-1 increased (P < 0.02) amounts of mRNA for IGF binding protein-3 and desmocollin II and tended (P < 0.08) to increase amounts of mRNA for Na/K ATPase and Bax. Moreover, IGF-1 treatment decreased (P < 0.05) steady-state amounts of transcripts for heat shock protein 70 and tended (P < 0.08) to reduce amounts of IGF-1 receptor mRNA. In conclusion, increased survival of embryos treated with IGF-1 does not appear due to effects on cell number, percent apoptosis, or cell allocation. Addition of IGF-1 to culture can, however, alter expression of several transcripts which may be important for embryo development and survival following transfer., ((c) 2007 Wiley-Liss, Inc.)

The present study investigated mRNA expression profiles of bovine oocytes and blastocysts by using a cross-species hybridization approach employing an array consisting of 15,529 human cDNAs as probe, thus enabling the identification of conserved genes during human and bovine preimplantation development. Our analysis revealed 419 genes that were expressed in both oocytes and blastocysts. The expression of 1,324 genes was detected exclusively in the blastocyst, in contrast to 164 in the oocyte including a significant number of novel genes. Genes indicative for transcriptional and translational control (ELAVL4, TACC3) were overexpressed in the oocyte, whereas cellular trafficking (SLC2A14, SLC1A3), proteasome (PSMA1, PSMB3), cell cycle (BUB3, CCNE1, GSPT1), and protein modification and turnover (TNK1, UBE3A) genes were found to be overexpressed in blastocysts. Transcripts implicated in chromatin remodeling were found in both oocytes (NASP, SMARCA2) and blastocysts (H2AFY, HDAC7A). The trophectodermal markers PSG2 and KRT18 were enriched 5- and 50-fold in the blastocyst. Pathway analysis revealed differential expression of genes involved in 107 distinct signaling and metabolic pathways. For example, phosphatidylinositol signaling and gluconeogenesis were prominent pathways identified in the blastocyst. Expression patterns in bovine and human blastocysts were to a large extent identical. This analysis compared the transcriptomes of bovine oocytes and blastocysts and provides a solid foundation for future studies on the first major differentiation events in blastocysts and identification of a set of markers indicative for regular mammalian development.

The effects of solid surface vitrification (SSV) on viability and parthenogenetic development of in vitro matured (IVM) porcine oocytes was investigated in the present study. Cumulus-free IVM porcine oocytes were subjected either to SSV or SSV combined with a cytochalasin B (CB) pre-treatment (SSV+CB) or all steps of SSV but without cooling (toxicity control=TC; toxicity control with CB pre-treatment=TC+CB). Oocyte viability was evaluated by plasma membrane integrity and esterase activity measured by a combined staining with fluorescein diacetate, propidium iodide and Hoechst 33342. Surviving oocytes were parthenogenetically activated then cultured in vitro (IVC) for 6 days. The proportion of live oocytes after vitrification was significantly lower than that of the TC, TC+CB and the control groups, regardless of the CB pre-treatment. Treatment of oocytes with cryoprotectants did not decrease the rates of surviving oocytes. After activation of oocytes, the proportion of cleaved embryos was significantly higher in the SSV+CB (P<0.05) than that of the SSV group. Nevertheless, significantly more oocytes cleaved (P<0.05) in the TC, TC+CB and the control groups. On Day 6, the rate of blastocysts in the SSV and SSV+CB groups did not differ significantly. The number of oocytes developing to blastocyst and the mean number of blastomeres per embryo were significantly higher (P<0.05) in the TC, TC+CB and the control compared with that of the SSV and SSV+CB groups. To our knowledge, this is the first report on parthenogenetic development to blastocysts of porcine oocytes vitrified at the metaphase-II stage. Results indicate that the high concentrations of cryoprotectants were not harmful for in vitro development, and that CB pre-treatment may increase survival and development of SSV vitrified porcine oocytes.

The goal of this study was to define conditions for the successful isolation of embryonic stem cells from bovine blastocysts. Expression of the Pit-Oct-Unc (POU) transcription factor Oct4 was employed to monitor the pluripotent status of cultured cells. No expression of the previously identified bovine Oct4 pseudogene was found, and transcription of the Oct4 ortholog correlated with the proliferative potential of bovine ICM derived cells. Two methods to isolate pluripotent inner cell mass were compared; 90% of trypsin isolated ICMs formed growing cultures, whereas only 12%-23% of the ICMs isolated by immunosurgery attached and grew. Colony formation from complete blastocysts was 55%. The bovine ICM derived cells could be grown for 4-7 passages. However, Oct4 transcripts were only present in the primary cultures, indicating that the initial culture period of bovine ICM derived cells is critical and needs to be optimized to yield true ES cells. In contrast to bovine ICMs, murine ICMs yielded rapidly growing cells, which proliferated for more than 60 passages., ((c) 2005 Wiley-Liss, Inc.)

The expression of nucleolar-related proteins was studied as an indirect marker of the ribosomal RNA (rRNA) gene activation in porcine embryos up to the blastocyst stage produced in vivo and in vitro. A group of the in vivo-developed embryos were cultured with alpha-amanitin to block the de novo embryonic mRNA transcription. Localization of proteins involved in the rRNA transcription (upstream binding factor [UBF], topoisomerase I, RNA polymerase I [RNA Pol I], and the RNA Pol I-associated factor PAF53) and processing (fibrillarin, nucleophosmin, and nucleolin) was assessed by immunocytochemistry and confocal laser-scanning microscopy, and mRNA expression was determined by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). These findings were correlated with ultrastructural data and autoradiography following 20-min [3H]uridine incubation. Additionally, expression of the pocket proteins pRb and p130, which are involved in cell-cycle regulation, was assessed by semiquantitative RT-PCR up to the blastocyst stage. Toward the end of third cell cycle, the nuclei in non-alpha-amanitin-treated, in vivo-produced embryos displayed different stages of transformation of the nuclear precursor bodies (NPBs) into fibrillogranular nucleoli associated with autoradiographic labeling. However, on culture with alpha-amanitin, NPBs were not transformed into a fibrillogranular nucleolus during this cell cycle, demonstrating that embryonic nucleogenesis requires de novo mRNA transcription. Moreover, immunolocalization of RNA Pol I, but not of UBF, and the mRNA expression of PAF53 and UBF were significantly reduced or absent after culture with alpha-amanitin, indicating that RNA Pol I, PAF53, and presumably, UBF are derived from de novo embryonic transcription. Embryonic genomic activation was delayed in porcine embryos produced in vitro compared to the in vivo-derived counterparts with respect to mRNAs encoding PAF53 and UBF. Moreover, differences existed in the mRNA expression patterns of pRb between in vivo- and in vitro-developed embryos. These findings show, to our knowledge for the first time, a nucleolus-related gene expression in the preimplantation porcine embryo, and they highlight the differences in quality between in vivo and in vitro-produced embryos.

The large offspring syndrome (LOS) is observed in bovine and ovine offspring following transfer of in vitro-produced (IVP) or cloned embryos and is characterized by a multitude of pathologic changes, of which extended gestation length and increased birthweight are predominant features. In the present study, we used bovine blastocysts to analyze cellular parameters, i.e., the number of cells in Day 7 blastocysts and the size of Day 12 elongating blastocysts, and molecular parameters, i.e., the relative abundance of developmentally important genes: glucose transporter (Glut) 1, Glut-2, Glut-3, Glut-4, heat shock protein (Hsp) 70.1, Cu/Zn-superoxide dismutase (SOD), histone H4.1, basic fibroblast growth factor (bFGF), insulin-like growth factor (IGF) I receptor (R), and IGFII-R. Some blastocysts were produced by in vitro maturation and fertilization followed by in vitro culture in synthetic oviduct fluid medium supplemented with BSA or human serum or by in vivo culture in the sheep oviduct. Other blastocysts were derived in vivo from the uterine horns of superovulated donors. The findings made in the early embryos were related to a representative number of calves obtained from each production system and from artificial insemination (AI). In vitro culture of bovine embryos in the presence of high concentrations of serum or BSA significantly increased the number of cells in Day 7 blastocysts, the size of blastocysts on Day 12, and the relative abundance of the transcripts for Hsp70.1, Cu/Zn-SOD, Glut-3, Glut-4, bFGF, and IGFI-R when compared with embryos from the in vivo production groups. Birthweights of calves derived from IVP embryos were significantly higher than those of calves derived from sheep oviduct culture, superovulation, or AI. The results support the hypothesis that persistence of early deviations in development is causally involved in the incidence of LOS, in particular in increased birthweights. The cellular and molecular parameters analyzed in this study can be considered early markers of LOS in cattle.

High cAMP levels during in vitro maturation (IVM) have been related to improved blastocyst yields. Here, we employed the cAMP/cGMP modulators, forskolin, IBMX, and cilostamide, during IVM to unravel the role of high cAMP in early embryonic development produced from prepubertal and adult bovine oocytes. Oocytes were collected via transvaginal aspiration and randomly assigned to three experimental groups: TCM24 (24h IVM/control), cAMP30 (2h pre-IVM (forskolin-IBMX), 30h IVM-cilostamide), and DMSO30 (Dimethyl Sulfoxide/vehicle control). After IVM, oocytes were fertilized in vitro and zygotes were cultured in vitro to blastocysts. Meiotic progression, cAMP levels, mRNA abundance of selected genes and DNA methylation were evaluated in oocytes. Blastocysts were used for gene expression or DNA methylation analyses. Blastocysts from the cAMP30 groups were transferred to recipients. The cAMP elevation delayed meiotic progression, but developmental rates were not increased. In immature oocytes, mRNA abundance of PRKACA was higher for cAMP30 protocol and no differences were found for PDE3A, SMAD2, ZAR1, PRDX1 and SLC2A8. EGR1 gene was up-regulated in prepubertal cAMP30 immature oocytes and down-regulated in blastocysts from all in vitro treatments. A similar gene expression profile was observed for DNMT3b, BCL2L1, PRDX1 and SLC2A8 in blastocysts. Satellite DNA methylation profiles were different between prepubertal and adult oocytes and blastocysts derived from the TCM24 and DMSO30 groups. Blastocysts obtained from prepubertal and adult oocytes in the cAMP30 treatment displayed normal methylation profiles and produced offspring. These data indicate that cAMP regulates IVM in prepubertal and adult oocytes in a similar manner, with impact on the establishment of epigenetic marks and acquisition of full developmental competency. [ABSTRACT FROM AUTHOR]

Pluripotent cells are unique because of their ability to differentiate into the cell lineages forming the entire organism. True pluripotent stem cells with germ line contribution have been reported for mice and rats. Human pluripotent cells share numerous features of pluripotentiality, but confirmation of their in vivo capacity for germ line contribution is impossible due to ethical and legal restrictions. Progress toward derivation of embryonic stem cells from domestic species has been made, but the derived cells were not able to produce germ line chimeras and thus are termed embryonic stem-like cells. However, domestic animals, in particular the domestic pig (Sus scrofa), are excellent large animals models, in which the clinical potential of stem cell therapies can be studied. Reprogramming technologies for somatic cells, including somatic cell nuclear transfer, cell fusion, in vitro culture in the presence of cell extracts, in vitro conversion of adult unipotent spermatogonial stem cells into germ line derived pluripotent stem cells, and transduction with reprogramming factors have been developed with the goal of obtaining pluripotent, germ line competent stem cells from domestic animals. This review summarizes the present state of the art in the derivation and maintenance of pluripotent stem cells in domestic animals. [ABSTRACT FROM AUTHOR]

Cryopreservation of in vitro produced bovine embryos is associated with significantly reduced survival rates, mainly due to insufficient quality of the embryos. Caffeine supplementation during IVM has been used to delay meiotic resumption and concomitantly also increased embryo quality. Here, we investigated the influence of pre-IVM with caffeine on oocyte maturation, intraoocyte cAMP concentration, developmental competence after IVF, and blastocyst cryotolerance. Oocytes were obtained by slicing of ovaries and were submitted to either 2 hours culture before IVM with or without caffeine (0, 1, 5, 10, 20, 30 mM), or standard IVM (no pre-IVM). Oocytes were in vitro matured and fertilized and zygotes were cultured under standard in vitro conditions until Day 8. Expanded blastocysts derived from either standard control or the 10-mM caffeine treatments were submitted to vitrification. Caffeine delayed meiotic resumption after 9-hour IVM in a concentration-dependent manner. The cAMP levels were similar before and after IVM. Matured oocytes, cleavage, and blastocyst rates were reduced in the 30-mM caffeine concentration and were similar among the other treatment groups. Number and proportion of inner cell mass and trophectoderm cells in blastocysts did not differ among treatments. Forty-eight hours after thawing, hatching rates were higher in the 10-mM caffeine group (73.8%) compared with the standard control (59.7%). Reexpansion rates and total number of cells after 48 hours were similar in both treatments. The ratio of live/total cells was higher in the caffeine treatment. These results suggest that caffeine supplementation before IVM delayed meiotic resumption and improved blastocyst quality shown in higher cryotolerance. [ABSTRACT FROM AUTHOR]