scholarly journals 45PRODUCTION OF CHIMERIC TRANSCHROMOSOMIC CALVES

2004 ◽  
Vol 16 (2) ◽  
pp. 144
Author(s):  
P. Kasinathan ◽  
M.F. Nichols ◽  
J.E. Griffin ◽  
J.M. Robl
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Peripheral Blood Lymphocytes ◽  
Blastocyst Stage ◽  
Human Artificial Chromosome ◽  
Fish Analysis ◽  
Blastocyst Development ◽  
Cell Stage ◽  
Fetal Fibroblasts

Chimeras have been used for investigating fundamental aspects of early embryonic development, and differentiation, and for introducing foreign genes into mammals (Robertson et al., 1986 Nature 323, 445–448; Cibelli et al., 1998 Science 280, 1256–1258). The main objective of this study was to determine if the transfer of blastomeres from in vitro-produced (IVP) embryos into cloned, transchromosomic embryos improved the efficiency of producing transchromosomic calves. Cloned embryos were produced using in vitro-matured bovine oocytes and bovine fetal fibroblasts containing a human artificial chromosome (HAC) (Kuroiwa et al., 2002 Nat Biotechnol 20, 889–894). IVP embryos were produced using standard procedures and blastomeres were harvested at the 8–16 cell stage by removing the zona pellucida with protease. Cloned embryos were randomly divided on Day 4 into two groups. One group received 3–4 IVP blastomeres while a second group served as a control (nonmanipulated cloned embryos). After transferring the blastomeres, the chimeric and cloned embryos were placed in culture (Kasinathan et al., 2001 Biol. Reprod. 64, 1487–1493) and on Day 7 development to the blastocyst stage was evaluated. Grades 1 and 2 embryos were transferred; two each per synchronized recipient. Pregnancy maintenance, calving, and calf survival were evaluated in both groups. Presence of a HAC in live calves was evaluated in both fibroblasts and peripheral blood lymphocytes (PBLs) using FISH analysis. Embryo development to the blastocyst stage, maintenance of pregnancy and number of calves born were analyzed using Chi-square. There were no differences in the rate of blastocyst development at day 7 or establishment of pregnancy at 40d (P>0.05). However, pregnancy rate at 120d, and number of calves that developed to term and were alive at birth (chimera 14/54 and clone 4/90), and at 1 month of age (chimera 13/54 and clone 1/90) were lower (P<0.01) for cloned embryos. The proportion of cells containing an HAC in PBLs, was higher in cloned calves (100%) compared to chimeric calves (26%). The HAC retension rates in PBLs in HAC-positive chimeric and cloned calves were 84% and 95%, respectively. These data indicate that, although the proportion of calves retaining an HAC was lower in chimeras compared to clones, more HAC-positive calves were produced in the chimeric treatment from fewer cloned embryos. We speculate that higher rates of development in the chimeras may be related to the normality of the placenta. Future studies will be required to determine the contribution of the IVP blastomeres to both the inner cell mass and trophectoderm. Therefore, a chimeric approach may be useful for improving the efficiency of producing cloned transchromosomic calves.

Potential of two-cell mouse embryos to develop to term despite partial damage after cryopreservation

1995 ◽  
Vol 29 (3) ◽  
pp. 320-326 ◽  
Author(s):  
Th. Rülicke ◽  
P. Autenried
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Blastocyst Stage ◽  
Mouse Embryos ◽  
Cell Stage ◽  
Freeze Thaw ◽  
Foster Mothers ◽  
Vital Stains ◽  
Partial Damage

Approximately 18% of cryopreserved 2-cell mouse embryos of 26 different batches showed various degrees of morphological damage after the freeze-thaw process. Normal and damaged morphology were assessed by light microscopy and the ability of an embryo to develop in vitro to a blastocyst, or to develop to term, after transfer to foster mothers. Using vital stains such as Fluorescein-diacetate (FDA) and 4',6-Diamidino-2-Phenylindole (DAPI) it was found that in approximately 82% of the cases, both of the 2 blastomeres of the cryopreserved embryos survived the freeze-thaw process; in 10% only one cell survived the process; and in 8% none survived. Normally, only intact 2-cell embryos are considered for transfer. Here it was shown that over 60% of the partially damaged embryos developed in vitro to the blastocyst stage and, of those, 26% developed to term after transfer to suitable foster mothers. Although the inner cell mass (ICM) appeared to remain smaller during culture after the transfer of partially damaged 2-cell stage embryos, no difference during gestation period was found compared with intact embryos.

53 TARGETED SCREEN FOR AMINO ACIDS THAT REGULATE BOVINE INNER CELL MASS DEVELOPMENT

2016 ◽  
Vol 28 (2) ◽  
pp. 156
Author(s):  
V. Najafzadeh ◽  
R. Martinus ◽  
B. Oback
Keyword(s):  
Inner Cell Mass ◽  
Statistical Significance ◽  
Cell Mass ◽  
Positive Control ◽  
Drop Out ◽  
Blastocyst Development ◽  
Acetyl Coa ◽  
Cell Numbers ◽  
Fetal Fibroblasts

Pluripotency relies on species-specific amino acid (AA) metabolism. In the mouse, inner cell mass (ICM) and ICM-derived pluripotent stem cells (PSCs) need threonine, which is catabolized by threonine dehydrogenase (TDH) into acetyl–CoA and glycine. Depleting (Δ) the culture medium of threonine (ΔT) or blocking TDH activity induces PSC death. By contrast, human PSCs do not survive without lysine (ΔK), leucine (ΔL), or methionine (ΔM). Since isolated bovine PSCs cannot be propagated in vitro, we screened for AAs that selectively support pluripotent ICM cells in intact bovine embryos. Five days (D5) post-IVF, embryos were transferred into glutamine-free synthetic oviduct fluid (gSOF) with Eagle’s nonessential (NE) and essential (E) AAs (gSOF_AA) plus BSA. Embryos were individually cultured until D8 under different conditions. Statistical significance was determined using Fisher’s exact test for blastocyst development (morphological grading to IETS standard) and t-tests for cell numbers (differential stain) and gene expression (quantitative or qPCR). Removal of BSA reduced grade 1–3 blastocyst (B1–3) development (37% v. 25%, n = 3; P < 0.001). Depleting NEAAs from gSOF_AA did not significantly decrease B1–3, but depleting all 12 EAAs did (25% v. 8%, n = 6; P < 0.001). Because ΔEAA was most effective, we focused on this. Experiments were conducted in gSOF+NEAA and compared with gSOF_AA as a positive control (n = 2–6 replicates). One (ΔT, ΔM), two (ΔMT, ΔCM, ΔCT; ΔIL, ΔIK, ΔKL), three (ΔCMT, ΔIKL), or six (ΔHPRVWY) EAA drop-out did not affect blastocyst formation, even when NEAAs were also removed for ΔT and ΔM groups (n = 3). However, depleting another six (ΔCIKLMT), nine (+CMT, +IKL), or eleven EAAs (+T, +M) increasingly compromised B1–3 (P < 0.05). Because no clear EAA candidates emerged from the screen, we focused on TDH. TDH mRNA was present at similar levels in microsurgically isolated (by microblade) trophectoderm (TE) and chemically isolated (by Triton X-100) ICM, but undetectable in five adult tissues. Despite ΔT medium showing no effect, exposure to the TDH inhibitor QC1 (50 µM) reduced B1–3 and B1–2 compared with a dimethylsulfoxide (DMSO) solvent control (25% v. 37% and 8% v. 19%, n = 8; P < 0.005). ICM and TE cell numbers were equally reduced in QC1 v. DMSO-treated blastocysts (10 v. 19 and 37 v. 67 with N = 21 and N = 29 embryos, respectively, n = 3; P < 0.005). Yet TDH, hypoblast (PDGRFα), epiblast (NANOG, FGF4, SOX2), and trophoblast (CDX2, KRT8) markers were not consistently affected by QC1. We next applied 3-hydroxynorvaline (3-HNV), which TDH hydrolyses into glycine and propionyl-CoA instead of acetyl-CoA. Compared with solvent controls, 3-HNV (300 µM) killed all embryos and bovine fetal fibroblasts within 3 days in ΔT medium. This toxic effect was fully rescued by >10-fold T-supplementation. Thus, 3-HNV protein incorporation, rather than acetyl-CoA reduction, may nonspecifically impair cellular function. In summary, we found that bovine ICM formation did not specifically depend on metabolizing threonine or any other single EAA. Research was supported by AgResearch Core Funding.

scholarly journals Development of Preimplantation Mouse Embryos in vivo and in vitro

1982 ◽  
Vol 35 (2) ◽  
pp. 187 ◽  
Author(s):  
GM Harlow ◽  
P Quinn
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Culture Conditions ◽  
Cell Stage ◽  
Preimplantation Mouse ◽  
Development In Vitro

The culture conditions for the development in vitro of (C57BL/6 X CBA) F2 hybrid two-cell embryos to the blastocyst stage have been optimized. Commercially available pre-sterile disposable plastic culture dishes supported more reliable development than re-usable washed glass tubes. The presence of an oil layer reduced the variability in development. An average of 85 % of blastocysts developed from hybrid two-cell embryos cultured in drops of Whitten's medium under oil in plastic culture dishes in an atmosphere of 5% O2 : 5% CO2 : 90% N2 ? The time taken for the total cell number to double in embryos developing in vivo was 10 h, and in cultured embryos 17 h. Embryos cultured in vitro from the two-cell stage to blastocyst stage were retarded by 18-24 h in comparison with those remaining in vivo. Day-4 blastocysts in vivo contained 25-70 cells (mean 50) with 7-28 (mean 16) of these in the inner cell mass. Cultured blastocysts contained 19-73 cells (mean 44) with 8-34 (mean 19) of these in the inner cell mass. In the uterine environment, inner-cell-mass blastomeres divided at a faster rate than trophectoderm blastomeres and it is suggested that a long cell cycle is associated with terminal differentiation. Although cultured blastocysts and inner cell masses contained the same number of cells as blastocysts and inner cell masses in vivo, the rate of cell division in cultured inner cell masses was markedly reduced.

scholarly journals CARM1 is heterogeneous in mouse four-cell embryo and important to blastocyst development

Reproduction ◽  
2020 ◽  
Vol 159 (1) ◽  
pp. 91-104
Author(s):  
Hongzheng Sun ◽  
Jianmin Su ◽  
Teng Wu ◽  
Fengyu Wang ◽  
Jian Kang ◽  
...  
Keyword(s):  
Cellular Proliferation ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Blastocyst Stage ◽  
Type I ◽  
Blastocyst Development ◽  
Cell Stage ◽  
Arginine Methyltransferase ◽  
Heterogeneous Distribution ◽  
Arginine Residues

Coactivator-associated arginine methyltransferase 1 (CARM1) is a type I arginine methyltransferase that methylates the arginine residues of histone and nonhistone. Carm1 regulates various cellular processes, including transcriptional regulation, mRNA processing, cellular proliferation, and differentiation. Blastomeres with high Carm1 expression levels show cleavage tendency to inner cell mass (ICM) in mouse embryos. However, details about the factors for CARM1 distribution in mouse early embryos and the role of Carm1 in blastocyst development remain unclear. Here, the endonuclear distribution of CARM1 protein was heterogeneous between blastomeres from the late four-cell stage to the blastocyst stage. The heterogeneity of CARM1 distribution in blastomeres at the late four-cell stage was randomly obtained from two-cell stage embryos. From the four-cell stage to morula, CARM1 in individual blastomere remained heterogeneous. In the blastocyst stage, CARM1 protein level in ICM was much higher than that in trophoblast. We found that microRNA (miRNA) miR-181a is an important regulator for Carm1 distribution at the late four-cell stage. The ratio of heterogeneous embryos was reduced in all the embryos when miR-181a was inhibited. CARM1 inhibition reduced the level of symmetrical histone H3 arginine-26 dimethylation and impaired blastocyst development. Silencing Carm1 reduced cell number and increased cell apoptosis at the blastocyst stage. These results show a CARM1 heterogeneous distribution from the four-cell embryos to the blastocysts. miR-181a regulates the control of CARM1 heterogeneous distribution in the four-cell-stage embryos, and CARM1 is an important protein in regulating blastocyst development.

scholarly journals Biological differences between in vitro produced bovine embryos and parthenotes

Reproduction ◽  
2009 ◽  
Vol 137 (2) ◽  
pp. 285-295 ◽  
Author(s):  
Enrique Gómez ◽  
Alfonso Gutiérrez-Adán ◽  
Carmen Díez ◽  
Pablo Bermejo-Alvarez ◽  
Marta Muñoz ◽  
...  
Keyword(s):  
De Novo ◽  
Inner Cell Mass ◽  
Relative Proportion ◽  
Cell Mass ◽  
Blastocyst Stage ◽  
Bovine Embryos ◽  
Blastocyst Development ◽  
Cell Counts ◽  
Biological Differences

Parthenotes may represent an alternate ethical source of stem cells, once biological differences between parthenotes and embryos can be understood. In this study, we analyzed development, trophectoderm (TE) differentiation, apoptosis/necrosis, and ploidy in parthenotes andin vitroproduced bovine embryos. Subsequently, using real-time PCR, we analyzed the expression of genes expected to underlie the observed differences at the blastocyst stage.In vitromatured oocytes were either fertilized or activated with ionomycin +6-DMAP and cultured in simple medium. Parthenotes showed enhanced blastocyst development and diploidy and reduced TE cell counts. Apoptotic and necrotic indexes did not vary, but parthenotes evidenced a higher relative proportion of apoptotic cells between inner cell mass and TE. The pluripotence-relatedPOU5F1and the methylationDNMT3Agenes were downregulated in parthenotes. Among pregnancy recognition genes,TP-1was upregulated in parthenotes, whilePGRMC1andPLAC8did not change. Expression ofp66shcandBAX/BCL2ratio were higher, andp53lower, in parthenotes. Among metabolism genes,SLC2A1was downregulated, whileAKR1B1,PTGS2,H6PD, andTXNwere upregulated in parthenotes, andSLC2A5did not differ. Among genes involved in compaction/blastulation,GJA1was downregulated in parthenotes, but no differences were detected withinATP1A1andCDH1. Within parthenotes, the expression levels ofSLC2A1,TP-1, andH6PD, and possiblyAKR1B1, resemble patterns described in female embryos. The pro-apoptotic profile is more pronounced in parthenotes than in embryos, which may differ in their way to channel apoptotic stimuli, throughp66shcandp53respectively, and in their mechanisms to control pluripotency andde novomethylation.

scholarly journals Mitogenic and anti-apoptotic activity of insulin on bovine embryos produced in vitro

Reproduction ◽  
2003 ◽  
pp. 91-99 ◽  
Author(s):  
R Augustin ◽  
P Pocar ◽  
C Wrenzycki ◽  
H Niemann ◽  
B Fischer
Keyword(s):  
Glucose Transporter ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Blastocyst Stage ◽  
Tunel Assay ◽  
Preimplantation Embryos ◽  
Blastocyst Development ◽  
Embryo Survival ◽  
Apoptotic Activity

Insulin improves development of mammalian preimplantation embryos and, in addition to the regulation of glucose transport, it exerts mitogenic and anti-apoptotic activities. The expression of glucose transporters (Glut) mediating the uptake of this essential energy substrate is critical for embryo survival. An impaired expression of Glut leads to an increase in apoptosis at the blastocyst stage and involves Bax. The various effects of insulin were unravelled by supplementing the in vitro culture medium with insulin (1.7 micromol l(-1)) and (i) the rates of cleavage and blastocyst development were recorded; (ii) mitogenic activity was studied by determining the total number of blastocyst cells and the ratio between trophectoderm and inner cell mass (ICM) cells; (iii) the frequency of apoptosis in blastocysts was determined by the TdT-mediated duTP nick-end labelling (TUNEL) assay and by quantification of the relative amounts of mRNA for Bax and Bcl-XL; and (iv) expression for Glut1, Glut3 and Glut8 transcripts was compared between embryos cultured in the presence or absence of insulin. Insulin increased rates of cleavage (81.2+/-2.2 (control) to 86.0+/-2.5) and blastocyst development (24.7+/-1.9 to 31.3+/-1.2), and number of blastocyst cells (123.7+/-6.0 to 146.3+/-6.6); the increase in the number of blastocyst cells was due to a significantly higher number of trophectoderm cells (82.3+/-5.0 versus 100.3+/-5.5). Blastocysts derived from cultures supplemented with insulin showed a significant decrease in apoptosis as determined by the TUNEL assay (14.8+/-0.9 to 12.2+/-0.7). No effects of insulin on the mRNA expression of Glut isoforms and Bax and Bcl-XL were found. These results demonstrate that the mitogenic and anti-apoptotic effects of insulin on bovine preimplantation embryos did not correlate with changes in the amounts of mRNA for the glucose transporter isoforms Glut1, -3 and -8, or transcripts for Bax and Bcl-XL.

218 DEVELOPMENT OF EVENLY AND UNEVENLY CLEAVED TWO-CELL PORCINE EMBRYOS

2008 ◽  
Vol 20 (1) ◽  
pp. 188
Author(s):  
D. N. Q. Thanh ◽  
K. Kikuchi ◽  
T. Somfai ◽  
M. Ozawa ◽  
M. Nakai ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Great Influence ◽  
Total Cell ◽  
Blastocyst Development ◽  
Cell Stage ◽  
Cell Numbers ◽  
Blastocyst Rate ◽  
Further Development

Mammalian eggs are so microlecithal that the embryos would be expected to divide in unison and that each division would lead to 2 equal blastomeres, which are believed to have a greater competence for further development than zygotes with unequal cleavage. However, some studies have shown that uneven blastomere size commonly occurs from the very first division in mammals, and it seems to be concerned with the generation of the first cell lineages of the blastocyst cells: trophectoderm and the inner cell mass (Gueth-Hallonet and Maro 1992 Trends Genet. 8, 274–279). In our study, we produced porcine embryos in vitro (Kikuchi et al. 2002 Biol. Reprod. 66, 1031–1041), and newly formed 2-cell embryos were collected. Based on the timing of the first cleavage (30 or 36 h after insemination), the cleavage pattern (E: equal; U: unequal) and the presence or absence of a second cleavage (+ or –) within the first 2 days of IVC was classified into groups: 30E(–), 30E(+), 30U(–), 30U(+), 36E(–), 36E(+), 36U(–), or 36U(+). There was no difference between the 30E and 30U groups in proportions of the 2-cell stage, which had a nucleus in both blastomeres (99.0 � 0.8% and 91.4 � 3.6%, respectively) or between the 36E and 36U groups (98.2 � 1.1% and 88.0 � 7.2%, respectively). Comparison of further development between the 30E and 30U groups showed that there was no difference in blastocyst rates (70.7 � 5.7% and 61.7 � 7.8%, respectively) and total cell numbers (39.1 � 2.1 and 31.7 � 2.3, respectively). Although the blastocyst rate in the 36E group (37.3 � 6.7%) was significantly higher (P < 0.05) than that of the 36U group (12.0 � 5.1%), the total cell number was not different (26.3 � 5.5 and 25.3 � 5.2, respectively). The timing of the first division, however, had a great influence on further development of the embryos; the 30-h cleaved embryos had a greater rate of blastocyst development (68.2 � 6.3%) than did the 36-h embryos (28.2 � 4.8%, P < 0.01 by ANOVA). The cell numbers of blastocysts derived from 30-h cleaved embryos (37.2 � 2.6) were significantly higher than those of the 36-h embryos (26.2 � 2.3, P < 0.01) as well. Two-cell embryos that were newly formed at 30 h and underwent the next cleavage within the first 2 days of IVC (30 + group) had a higher blastocyst rate (74.8 � 7.0%) and greater cell numbers (40.6 � 2.6) than those not showing a second division during this period (30– group; 46.8 � 5.0% and 19.9 � 2.2, respectively). In contrast, for embryos showing the first cleavage at 36 h of insemination, the presence of the next cleavage within 2 days after the first cleavage did not have any effect on embryonic development. These results suggest that the developmental ability of porcine embryos was influenced by the timing and shape of the first cleavage and by the subsequent occurrence of the second cleavage.

scholarly journals The role of Trp53 in the mouse embryonic response to DNA damage

2019 ◽  
Vol 25 (7) ◽  
pp. 397-407
Author(s):  
Yvonne Wilson ◽  
Ian D Morris ◽  
Susan J Kimber ◽  
Daniel R Brison
Keyword(s):  
Dna Damage ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Blastocyst Stage ◽  
Human Embryos ◽  
Cell Stage ◽  
X Irradiation

Abstract Apoptosis occurs primarily in the blastocyst inner cell mass, cells of which go on to form the foetus. Apoptosis is likely to play a role in ensuring the genetic integrity of the foetus, yet little is known about its regulation. In this study, the role of the mouse gene, transformation-related protein 53 (Trp53) in the response of embryos to in vitro culture and environmentally induced DNA damage was investigated using embryos from a Trp53 knockout mouse model. In vivo-derived blastocysts were compared to control embryos X-irradiated at the two-cell stage and cultured to Day 5. An analysis of DNA by comet assay demonstrated that 1.5 Gy X-irradiation directly induced damage in cultured two-cell mouse embryos; this was correlated with retarded development to blastocyst stage and increased apoptosis at the blastocyst stage but not prior to this. Trp53 null embryos developed to blastocysts at a higher frequency and with higher cell numbers than wild-type embryos. Trp53 also mediates apoptosis in conditions of low levels of DNA damage, in vivo or in vitro in the absence of irradiation. However, following DNA damage induced by X-irradiation, apoptosis is induced by Trp53 independent as well as dependent mechanisms. These data suggest that Trp53 and apoptosis play important roles in normal mouse embryonic development both in vitro and in vivo and in response to DNA damage. Therefore, clinical ART practices that alter apoptosis in human embryos and/or select embryos for transfer, which potentially lack a functional Trp53 gene, need to be carefully considered.

scholarly journals Totipotency of mouse zygotes extends to single blastomeres of embryos at the four-cell stage

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Marino Maemura ◽  
Hiroaki Taketsuru ◽  
Yuki Nakajima ◽  
Ruiqi Shao ◽  
Ayaka Kakihara ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Mouse Embryos ◽  
Primitive Endoderm ◽  
Cell Stage ◽  
Supportive Environment ◽  
Mouse Zygotes ◽  
Single Blastomeres ◽  
Multicellular Organisms

AbstractIn multicellular organisms, oocytes and sperm undergo fusion during fertilization and the resulting zygote gives rise to a new individual. The ability of zygotes to produce a fully formed individual from a single cell when placed in a supportive environment is known as totipotency. Given that totipotent cells are the source of all multicellular organisms, a better understanding of totipotency may have a wide-ranging impact on biology. The precise delineation of totipotent cells in mammals has remained elusive, however, although zygotes and single blastomeres of embryos at the two-cell stage have been thought to be the only totipotent cells in mice. We now show that a single blastomere of two- or four-cell mouse embryos can give rise to a fertile adult when placed in a uterus, even though blastomere isolation disturbs the transcriptome of derived embryos. Single blastomeres isolated from embryos at the eight-cell or morula stages and cultured in vitro manifested pronounced defects in the formation of epiblast and primitive endoderm by the inner cell mass and in the development of blastocysts, respectively. Our results thus indicate that totipotency of mouse zygotes extends to single blastomeres of embryos at the four-cell stage.

scholarly journals Cell lineage allocation in equine blastocysts produced in vitro under varying glucose concentrations

Reproduction ◽  
2015 ◽  
Vol 150 (1) ◽  
pp. 31-41 ◽  
Author(s):  
Young-Ho Choi ◽  
Pablo Ross ◽  
Isabel C Velez ◽  
B Macías-García ◽  
Fernando L Riera ◽  
...  
Keyword(s):  
High Glucose ◽  
Culture Medium ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Lineage ◽  
Primitive Endoderm ◽  
Blastocyst Development ◽  
Embryo Culture Medium ◽  
Effect Of Glucose

Equine embryos developin vitroin the presence of high glucose concentrations, but little is known about their requirements for development. We evaluated the effect of glucose concentrations in medium on blastocyst development after ICSI. In experiment 1, there were no significant differences in rates of blastocyst formation among embryos cultured in our standard medium (DMEM/F-12), which contained >16 mM glucose, and those cultured in a minimal-glucose embryo culture medium (<1 mM; Global medium, GB), with either 0 added glucose for the first 5 days, then 20 mM (0-20) or 20 mM for the entire culture period (20-20). In experiment 2, there were no significant differences in the rates of blastocyst development (31–46%) for embryos cultured in four glucose treatments in GB (0-10, 0-20, 5-10, or 5-20). Blastocysts were evaluated by immunofluorescence for lineage-specific markers. All cells stained positively forPOU5F1. An inner cluster of cells was identified that included presumptive primitive endoderm cells (GATA6-positive) and presumptive epiblast (EPI) cells. The 5-20 treatment resulted in a significantly lower number of presumptive EPI-lineage cells than the 0-20 treatment did.GATA6-positive cells appeared to be allocated to the primitive endoderm independent of the formation of an inner cell mass, as was previously hypothesized for equine embryos. These data demonstrate that equine blastocyst development is not dependent on high glucose concentrations during early culture; rather, environmental glucose may affect cell allocation. They also present the first analysis of cell lineage allocation inin vitro-fertilized equine blastocysts. These findings expand our understanding of the factors that affect embryo development in the horse.

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