In Vitro Production System Research Articles

Micro-vibration results in vitro-derived bovine blastocysts with greater cryotolerance, epigenetic abnormalities, and a massive transcriptional change

Much effort has been employed to improve the quality of embryos obtained by in vitro production (IVP) given the relevance of this technology to current livestock systems. In this context, dynamic IVP systems have proved beneficial to the embryo once they mimic fluid flows and mechanical forces resulting from the movement of ciliated cells and muscle contraction in the reproductive tract. In the present study, we sought to confirm these initial findings as well as assess potential molecular consequences to the embryo by applying micro-vibration (45 Hz for 5 s once per 60 min) during both oocyte maturation and embryo culture in cattle. As a result, micro-vibration led to lower incidence of apoptosis in blastocysts following vitrification-thawing. Further analyses revealed epigenetic and transcriptional changes in blastocysts derived from the micro-vibration treatment, with a total of 502 differentially expressed genes. Enrichment analyses linked differentially expressed genes to ‘Oxidative phosphorylation’, ‘Cytokine-cytokine receptor interaction’, and ‘Signaling pathways regulating pluripotency of stem cells’. Yet, a meta-analysis indicated that the transcriptional changes induced by micro-vibration were not toward that of in vivo-derived embryos. In conclusion, micro-vibration increases the cryoresistance of bovine embryos, but caution should be taken given the unclear consequences of epigenetic and transcriptional abnormalities induced by the treatment.

Simple and Efficient Chemically Defined In Vitro Maturation and Embryo Culture System for Bovine Embryos.

Supplementation of the culture media for in vitro production (IVP) of bovine embryos with fetal bovine serum (FBS) is associated with inconsistent outcomes. The present study sought to replace FBS and BSA by insulin-like growth factor 1 (IGF1), fibroblast growth factor 2 (FGF2) and epidermal growth factor (EGF). In Experiment 1, absence of FBS from maturation medium (MM) did not affect the rate of in vitro maturation, as assessed by the extrusion of the first polar body. However, when gonadotropins and FBS were removed from the MM, the maturation rate was significantly reduced even in the presence of growth factors. Therefore, gonadotropin-supplemented MM medium was established as the base medium for the defined maturation condition. In Experiment 2, the addition of growth factors to gonadotropin-supplemented MM medium supported similar maturation (~90%) compared to the undefined condition (FBS-carrying). In Experiment 3, the addition of growth factors to embryo culture medium showed similar in vitro competence compared to the undefined (FBS) control. In Experiment 4, completely defined conditions (absence of FBS and BSA during in vitro maturation and embryo culture) were tested. A higher cleavage was observed with FGF2 (86%) compared to EGF (77%) and the FBS control (77%), but similar blastocyst rates were observed for FGF2 (24%), EGF (19%) and the FBS control (25%). Embryo quality was similar among groups. Finally, post-thawing survival was higher for FGF2 (94%) compared to the FBS control (77%). Thus, we report a simple defined IVP system for bovine species that generates developmental outcomes and embryos of similar quality than those produced under conditions containing FBS.

In vitro production of buffalo embryos in Colombia – results of the addition of growth factors and antioxidants to defined culture medium

To increase the success rate of the in vitro production (IVP) system of embryos in buffalo, it is necessary to improve parameters. This field requires more research since its results are low compared to cattle. In this study carried out in Colombia, a modification of the methodology to produce buffalo IVP embryos is described, and 24 non-lactating females, aged 4 to 11 years old were included. Follicular wave was synchronised by ablation of the follicles within the ovary, seven days later all follicles were aspirated to obtain oocytes for maturation, fertilisation and embryo production. For all culture media used, animal protein was avoided. Culture media were supplemented with growth factors and other molecules associated with embryonic development. Special care was taken in adding supplements to protect the zygotes against the harmful effect of oxygen free radicals in the culture medium. Two aspiration series were performed 15 days apart. The average number of oocytes obtained by aspiration was 11.33, the number of blastocysts was 3.05, and the obtained blastocyst production rate (36.2%) was higher than literature reports for this species; we hypothesise that this increase is associated with the supplements added. It was possible to observe a female that produced 50 oocytes. Subsequent research is needed to evaluate the effect of the medium on embryo quality and pregnancy rates. A cost analysis of the new proposed medium is required, since all supplements must be imported and handled with a cold chain of up to -80oC, making it more expensive and restricting widespread use. Finally, it is shown that with three embryos per aspiration, at least one pregnancy can be successful after the aspiration, which is one of the objectives of the IVP system.

Supplementation of porcine in vitro maturation medium with FGF2, LIF, and IGF1 enhances cytoplasmic maturation in prepubertal gilts oocytes and improves embryo quality

In porcine in vitro production (IVP) systems, the use of oocytes derived from prepubertal gilts, whilst being commercially attractive, remains challenging due to their poor developmental competence following in vitro maturation (IVM). Follicular fluid contains important growth factors and plays a key role during oocyte maturation; therefore, it is a common supplementation for porcine IVM medium. However, follicular fluid contains many poorly characterized components, is batch variable, and its use raises biosecurity concerns. In an effort to design a defined IVM system, growth factors such as cytokines have been previously tested. These include leukaemia inhibitory factor (LIF), fibroblast growth factor 2 (FGF2), and insulin-like growth factor 1 (IGF1), the combination of which is termed 'FLI'. Here, using abattoir-derived oocytes in a well established porcine IVP system, we compared follicular fluid and FLI supplementation during both IVM and embryo culture to test the hypothesis that FLI can substitute for follicular fluid without compromising oocyte nuclear and cytoplasmic maturation. We demonstrate that in oocytes derived from prepubertal gilts, FLI supplementation enhances oocyte meiotic maturation and has a positive effect on the quality and developmental competence of embryos. Moreover, for the first time, we studied the effects of follicular fluid and FLI combined showing no synergistic effects.

PGE2 Supplementation of Oocyte Culture Media Improves the Developmental and Cryotolerance Performance of Bovine Blastocysts Derived From a Serum-Free in vitro Production System, Mirroring the Inner Cell Mass Transcriptome.

The culture media used throughout the in vitro production (IVP) of bovine embryos remain complex. The serum added to culture media in order to improve embryo development negatively impacts the cryotolerance of blastocysts. Periconceptional prostaglandin E2 (PGE2) signaling is known to exert prosurvival effects on in vitro-generated blastocysts. The purpose of the present study was to evaluate the effects on developmental and cryotolerance performance of a serum-free (SF) IVP system that included defined oocyte culture media supplemented or not with PGE2, versus serum-containing (SC) IVP. RNA-sequencing analysis was used to examine the gene expression of ICM derived under the different IVP conditions. We assessed the degree of cryotolerance of grade-I blastocysts during a three-day post-thaw culture by measuring survival and hatching rates, counting trophectoderm and inner cell mass (ICM) blastomere numbers. We also determined the proportion of ICM cells expressing octamer-binding transcription factor 4 protein (OCT4/POU5F1). We showed that grade-I blastocyst development rates under SF + PGE2 conditions were similar to those obtained under SC conditions, although the cleavage rate remained significantly lower. SC IVP conditions induced changes to ICM gene expression relative to several metabolic processes, catabolic activities, cell death and apoptosis. These alterations were associated with significantly higher levels of ICM cell death at day 7 post-fertilization, and lower survival and hatching rates after thawing. SF IVP conditions supplemented or not with PGE2 induced changes to ICM gene expression related to DNA replication, metabolism and double-strand break repair processes, and were associated with significantly larger ICM cell populations after thawing. SF + PGE2 IVP induced changes to ICM gene expression related to epigenetic regulation and were associated with a significantly higher proportion of ICM cells expressing OCT4. For the first time, our study thus offers a comprehensive analysis of the ICM transcriptome regulated by IVP culture conditions in terms of the cellular changes revealed during culture for three days after thawing.

120 High variation in prediction of developmental competence of bovine oocytes based on visual examination

The quality of an oocyte is often represented by its morphological characteristics. Stringent selection of bovine cumulus–oocyte complexes (COCs) for invitro embryo production (IVP) is regularly based on parameters such as colour and granulation of the ooplasm, density of the surrounding cumulus cells, brightness of the zona pellucida, among other less common parameters. The link between developmental competence and morphology of the COC has been demonstrated in literature in group culture system. However, this has not been yet examined in an individual invitro production system. This study investigates the ability of IVP researchers to predict whether an oocyte will develop into a blastocyst at Day 8 of invitro culture in an individual production system. A set of 29 pictures of immature bovine COCs was presented in duplicates to eight bovine IVP researchers with different institutional backgrounds. The observers were asked if they would select each oocyte for further IVP processing, assuming that only the oocytes with the highest developmental potential can proceed, and thus a stringent selection is warranted. These 29 immature oocytes were matured, fertilized, and cultured individually invitro (using conventional methods) so that their ability to develop into a blastocyst at Day 8 post-fertilization was known. The ability to reach the blastocyst stage was stated as the gold standard and compared with the answers of the observers using kappa statistics and sensitivity (Se) and specificity (Sp) tests. Kappa (κ) for interobserver agreement was κ=0.01 (Se=50%, Sp=53%; positive predictive value=14%, negative predictive value=87%) with 50% accuracy of prediction. The intraobserver agreement was κ=0.69, with κ=1 referring to 100% agreement (Se=87%, Sp=83%), with an accuracy of 85%. The ability of trained embryologists to predict the fate of oocytes’ developmental competence was accurate for only half of the presented oocytes. Additionally, the proportion of false positive (1 − Sp) and false negative (1−Se) predictions was equally distributed. The low positive predictive value might be due to the lower development rates in individual culture systems versus group culture systems, whereas observers are mainly trained to select oocytes for group culture. This study demonstrated the subjective nature of the oocyte selection process that is based on visual examination of COC morphology because interobserver agreement was almost nonexistent. Interpretation of the morphology within an observer remains consequent, represented by the high intraobserver agreement. Consequently, there is a need to develop a new model for the objective determination of oocyte quality for individual IVP. The implementation of machine learning algorithms could objectively enhance oocyte selection and artificial reproductive technologies in extension.

Importance of oil overlay for production of porcine embryos in vitro.

Technologies to edit the zygote genome have revolutionized biomedical research not only for the creation of animal models for the study of human disease but also for the generation of functional human cells and tissues through interspecies blastocyst complementation technology. The pig is the ideal species for these purposes due to its great similarity in anatomy and physiology to humans. Emerging biotechnologies require the use of oocytes and/or embryos of good quality, which might be obtained using in vitro production (IVP) techniques. However, the current porcine embryo IVP systems are still suboptimal and result in low monospermic fertilization and blastocyst formation rates and poor embryo quality. During recent years, intensive investigations have been performed to evaluate the influence of specific compounds on gametes and embryos and to avoid the use of undefined supplements (serum and serum derivate) in the incubation media. However, little consideration has been given to the use of the mineral oil (MO) to overlay incubation droplets, which, albeit being a routine component of the IVP systems, is a totally undefined and thus problematic product for the safety of gametes and embryos. In this review, we provide an overview on the advantages and disadvantages of using MO to cover the incubation media. We also review one important concern in IVP laboratories: the use of oils containing undetected contamination. Finally, we discuss the effects of different types of oils on the in vitro embryo production outcomes and the transfer of compounds from oil into the culture media.

Chlorogenic acid supplementation during in vitro maturation improves maturation, fertilization and developmental competence of porcine oocytes.

Chlorogenic acid (CGA) is a quinic acid conjugate of caffeic acid, and a phytochemical found in many fruits and beverages that acts as an antioxidant. The present study investigated the effects of CGA supplementation during in vitro maturation (IVM), on in vitro development of porcine oocytes, to improve the porcine in vitro production (IVP) system. Oocytes were matured either without (control) or with CGA (10, 50, 100 and 200μM). Subsequently, the matured oocytes were fertilized and cultured in vitro for 7day. The rates of maturation, fertilization and blastocyst formation of oocytes matured with 50μM CGA were significantly (p<.05) higher than those of the control oocytes. Hydrogen peroxide (H2 O2 ) is one of the reactive oxygen species and induces DNA damage in porcine oocytes. When oocytes were matured with 1mM H2 O2 to assess the protective effect of CGA, 50μM CGA supplementation improved the maturation rate and the proportion of DNA-fragmented nuclei in oocytes compared with control oocytes matured without CGA. Moreover, when oocytes were matured with either 50μM CGA (control) or caffeic acid (10, 50 and 100μM), the rates of maturation, fertilization and the blastocyst formation of oocytes matured with 50μM CGA were similar to those of oocytes matured with 10 and 50μM caffeic acid. Our results suggest that CGA has comparable effects to caffeic acid, and IVM with 50μM CGA is particularly beneficial to IVP of porcine embryos and protects oocytes from DNA damage induced by oxidative stress. Supplementation of CGA to the maturation medium has a potential to improve porcine IVP system.

Comprehensive cross production system assessment of the impact of in vitro microenvironment on the expression of messengers and long non-coding RNAs in the bovine blastocyst

In vitro production (IVP) of cattle embryos over the past two decades has revealed several negative impacts that have been attributed to the artificial microenvironment. Studies on embryos produced in vitro clearly point to aberrant gene expression levels. So far, the causal association between phenotype and measured gene expression has not led to substantial improvement of IVP systems. The aim of this study was to generate a unique dataset composed of microarray-derived relative transcript abundance values for blastocysts produced in ten in vitro systems differing primarily in culture medium formulation. Between-group comparisons determine the level of overall similarity among systems relative to in vivo reference embryos. The use of the dataset to contrast all in vitro treatments with the in vivo blastocysts pointed to a single common gene network. The 'boutique' array contained a panel of novel uncharacterized transcripts that were variably expressed depending on the medium in which the blastocysts were produced. These novel transcripts were differentially expressed in blastocysts even as carryover from conditions encountered 7 days earlier during oocyte maturation. All of the selected novel candidates thus expressed were from intergenic regions. The function of this long non-coding RNA remains unknown but clearly points to an additional level of complexity in early embryo development.

Development and Application of a Chemically Defined Medium for the In Vitro Production of Porcine Embryos

Recent advances in systems for in vitro production (IVP) of porcine embryos, including in vitro oocyte maturation, fertilization and embryo culture, have enabled us to generate viable embryos that can develop to full term after transfer into recipients. This technology is being applied now to developments in gamete/embryo biology and agriculture, as well as in producing cloned and genetically modified pigs. Chemically defined media for IVP of embryos are useful for a precise analysis of the physical action of substances on gametogenesis and early embryogenesis, because they eliminate undefined factors present in biological materials, such as serum or serum albumin. Use of a chemically defined medium also improves the reliability of media formulations, yields a higher reproducibility of results and ensures biosafety of culture media by eliminating protein preparations, which may be contaminated with pathogens. Therefore, it has certain advantages for research and for commercial purposes. We have recently developed a defined IVP system for porcine embryos using a single basic medium based on the composition of porcine oviductal fluid. This paper discusses the developmental ability and normality of porcine IVP embryos, and limitations and advancements in this system.

210 CO-CULTURE WITH AUTOLOGOUS CUMULUS CELLS SUPPORTS THE INDIVIDUAL DEVELOPMENT OF SINGLY IN VITRO-MATURED AND FERTILIZED BOVINE OOCYTES

The ability to produce embryos singly in vitro (in vitro production, IVP) would be a useful tool for many purposes. Without the interfering effects of other developing or degenerating oocytes or embryos, such an individual IVP system is the tool of choice for studies on oocyte quality and oocyte–embryo metabolism. Unfortunately, individual IVP in most cases leads to unsatisfactorily low blastocyst rates. Earlier work showed that individual culture of zygotes on a cumulus cell (CC) monolayer resulted in comparable numbers of good-quality embryos, as obtained following regular group culture (Goovaerts et al. 2009 Theriogenology 71, 729–738). However, co-culture with somatic cells is often criticised because of the undefined culture conditions and for sanitary reasons. In the cited study, CC for monolayer production were obtained from a different batch of ovaries. Our specific aim was to use CC from the zygote itself (autologous CC). Grade I COC (n = 660) were collected from slaughterhouse ovaries and randomly assigned to 2 treatments (5 replicates): a completely individual ‘single-oocyte’ IVP protocol, or routine group IVP as a control. Individual maturation (TCM-199 + 20% serum) and fertilization were performed in 20-μL droplets under oil in 24-well plates. Subsequently, each zygote was stripped and cultured in 20 μL of medium (SOF + 5% serum, 90% N2, 5% CO2, 5% O2), to which the autologous stripped CC were added. Group maturation and fertilization were carried out per 100 COC in 500 μL, whereas group culture was performed per 25 zygotes in 50-μL droplets under oil. Cleavage, blastocyst, and hatching rates were determined 2, 8, and 10 days post-fertilization, respectively. Possible effects of the individual and group cultures were evaluated with binary logistic regression (SPSS 15.0, SPSS Inc., Chicago, IL). No interactions between replicate and treatment were found (P &gt; 0.05). Although a blastocyst rate of 15.1% was obtained using single IVP, the general efficacy of the single-embryo production system was lower when compared with group culture (Table 1). In conclusion, although developmental competence was impaired using individual IVP, co-culture with autologous cumulus cells can be useful in specific experimental setups in which the influence of other oocytes or embryos or heterologous somatic cells is unacceptable. Table 1.Cleavage, blastocyst, and hatching rates after individual and group in vitro production (IVP)

Ovine (Ovis aries) blastula from an in vitro production system and isolation of primary embryonic stem cells

Livestock embryo production in in vitro systems has been highlighted due to the emergence of interest in embryo stem cells (ESC). ESC potency and their wide potential applications have been recognized in medicine, fundamental research fields and commercial markets due to ESC totipotency or pluripotency and self-renewal. Ovine ESC probably is a useful technical platform for transgenic livestock and animal cloning, but ESC lines have not yet been founded because of difficulties in ESC isolation and the lack of blastula materials. We have established an IVP (in vitro production) system in our laboratory, including in vitro maturation, in vitro fertilization and in vitro culture, to produce sheep blastula using fresh ovaries and testes collected from livestock production. This system can achieve rates of mature eggs and blastulas of 65 and 50% respectively, and can provide enough blastulas for ICM (inner cell mass) isolation. Furthermore, ESC-like clones were isolated from the ICM on ovine embryonic fibroblast (OEF) feeder cells and in ES-DMEM supplemented with the cell factors LIF and SCF, and these survived to the third passage, which was primarily identified by AKP staining and morphology. This work provides a basis for ovine ESC isolation and foundation of ESC lines.

277ENERGY REQUIREMENT DURING DEVELOPMENT TO THE BLASTOCYST STAGE OF PORCINE EMBRYOS PRODUCED IN VITRO

A successful in vitro production (IVP) of porcine blastocysts, which enables piglet production after transfer to recipients, was reported (Kikuchi et al., 2002 Biol. Reprod. 66, 1033–1041). Generally, in the IVP system, both glucose and glutamine as energy sources were included in vitro culture (IVC) medium from Day 2 (Day 0=the day of in vitro fertilization) until Day 6. However, the exact requirement of these substances for the development to the blastocyst stage of IVP embryos has not yet been clarified. The objective of the present study was to evaluate whether these two substances are necessary for embryonic development to the blastocyst stage in culture during the period. Porcine cumulus-oocyte complexes were matured for 46h and fertilized in vitro as reported by Kikuchi et al. (see above). After removal of cumulus cells and spermatozoa, the oocytes were cultured subsequently in NCSU-37 supplemented with pyruvate and lactate (IVC-PyrLac) for 2 days. Then they were cultured until Day 6 in other IVC medium prepared as follows (1–6); Basic IVC medium (BM) was a modified NCSU-37 consisting of 108.7mM NaCl, 4.8mM KCl, 1.7mM CaCl2, 1.2mMKH2PO4, 1.2mM MgSO4, 25.1mM NaHCO3 and 4mgmL−1 fatty acid-free BSA. Then one or more of the following energy sources were supplemented to BM;; (1) 12mM sorbitol (SigmaUltra), 5.55mM glucose (Wako special grade) and 1.0mM glutamine (Sigma) (NCSU-37/Gln+), (2) 19.2mM sorbitol and 1.0mM glutamine (IVC-Sorbitol/Gln+); (3) 19.2mM mannitol (SigmaUltra) and 1.0mM glutamine (IVC-Mannitol/Gln+), (4) 12mM sorbitol and 5.55mM glucose (NCSU-37/Gln−); 5) 19.2mM sorbitol (IVC-Sorbitol/Gln−); and 6) 19.2mM mannitol (IVC-Mannitol/Gln−). The osmolarity of these media was adjusted to 283–285 osmolg−1. All embryos were fixed as whole mounts, stained and evaluated. The rate of blastocysts in NCSU-37/Gln+ (26.8%) was significantly higher (P&amp;lt;0.05; by analysis of variance and Duncan’s multiple range test) than those in IVC-Sorbitol/Gln+, IVC-Mannitol/Gln+ and NCSU-37/Gln− (19.0%, 17.0% and 15.5%, respectively). A remarkable decrease in the rates in IVC-Sorbitol/Gln− and IVC-Mannitol/Gln− (P&amp;lt;0.05; 1.4% and 2.0%, respectively) was observed. The cell numbers of NCSU-37/Gln+, IVC-Sorbitol/Gln+, IVC-Mannitol/Gln+ and NCSU-37/Gln− (55.5, 52.0, 49.6 and 58.7, respectively) had a tendency to be higher than those of IVC-Sorbitol/Gln− and IVC-Mannitol/Gln− (38.0 and 35.2, respectively). These results confirm that the supplementation of maturation medium with at least one energy source (glucose or glutamine) promotes embryonic development in vitro to the blastocyst stage, that the combination of both sources improves the chance of the embryonic survival, and that porcine embryos do not utilize sorbitol or mannitol as an energy source. The importance of glucose and glutamine is suggested for the development to the blastocyst stage of porcine IVP embryos.

Successful piglet production after transfer of blastocysts produced by a modified in vitro system.

Porcine in vitro production (IVP) systems, including in vitro maturation (IVM) and in vitro fertilization (IVF) of oocytes and their subsequent in vitro culture (IVC), have been modified by many researchers, but are still at a low level because of a low developmental rate of embryos to the blastocyst stage and their poor qualities. Our objectives were to establish reliable IVP procedures for porcine blastocysts and to examine the ability of the blastocysts to develop to term after transfer to recipients. Porcine cumulus-oocyte complexes were matured in vitro under 5% O(2) or 20% O(2), fertilized in vitro under 5% O(2), and subsequently cultured under 5% O(2) in 1) IVC medium supplemented with glucose (IVC-Glu) from Day 0 (the day of IVF) to Day 6; 2) IVC-Glu from Days 0 to 2, then IVC medium supplemented with pyruvate and lactate (IVC-PyrLac) from Days 2 to 6; 3) IVC-PyrLac from Days 0 to 2, then IVC-Glu from Days 2 to 6; and 4) IVC-PyrLac from Days 0 to 6. There were no significant differences in blastocyst formation rates on Day 6 between the 5% O(2) and 20% O(2) conditions (19.9% and 14.0%, respectively). However, the quality of blastocysts, as evaluated by the total cell number, was better after IVM under 5% O(2) than under 20% O(2) (mean cell number, 43.5 and 37.8, respectively). When IVP embryos were cultured in IVC-PyrLac from Days 0 to 2 and subsequently in IVC-Glu from Days 2 to 6, the rate of blastocyst formation (25.3%) and cell number (48.7) were higher than the rates (5.8% to 18.1%) and numbers (35.4 to 37.1) with the IVC-Glu then IVC-Glu, the IVC-Glu then IVC-PyrLac, and the IVC-PyrLac then IVC-PyrLac regimens, respectively. We then prepared conditioned medium (CM) from culture of porcine oviductal epithelial cells for 2 days in IVC-PyrLac and evaluated its effect on development to the blastocyst stage. Cultivation in CM for the first 2 days, followed by IVC-Glu for a further 4 days, had a significantly greater effect in increasing the number of cells in the blastocyst (58.3) than did in IVC-PyrLac (48.4). Finally, we evaluated the ability of blastocysts, generated by IVM under 5% O(2) and IVC in CM, to develop to term. When Day 5 expanding blastocysts (mean cell number, 49.7) were transferred to an estrus-synchronized recipient (50 blastocysts per recipient), the recipient remained pregnant and farrowed eight normal piglets. Furthermore, when Day 6 expanded blastocysts (mean cell number, 80.2) were transferred to two estrus-synchronized recipients, both gilts remained pregnant and farrowed a total of 11 piglets. These results suggest that an excellent piglet production system can be established by using this modified IVP system, which produces high-quality porcine blastocysts. This system has advantages for the generation of cloned and transgenic pigs.

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.