scholarly journals Bovine blastocyst development depends on threonine catabolism

2018 ◽  
Author(s):  
V Najafzadeh ◽  
H Henderson ◽  
R Martinus ◽  
B Oback
Keyword(s):  
Amino Acid ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Essential Amino Acids ◽  
Continuous Exposure ◽  
Blastocyst Development ◽  
Amino Acid Requirements ◽  
Species Specific ◽  
Bovine Blastocyst

ABSTRACTIncreasing evidence suggests that pluripotency is a metabolically specialised state. In mouse, inner cell mass (ICM) cells and ICM-derived pluripotent stem cells (PSCs) critically depend on catabolising the amino acid threonine, while human PSCs require leucine, lysine, methionine or tryptophan. However, little is known about the specific amino acid requirements of putative pluripotent cells in bovine. We selectively depleted candidate essential amino acids (EAAs) from individually cultured bovine embryos to study their role in blastocyst development. Depleting one (-T, -M), two (-MT, -CM, -CT, -IL, -IK, -KL) or three (-CMT, -IKL) EAAs from chemically defined protein-free culture medium did not affect the morula-to-blastocyst transition from day five (D5) to D8 in vitro. By contrast, removing six (-CIKLMT, -FHRYVW), nine (+CMT, +IKL), eleven EAAs (+T, +M) or all twelve EAAs increasingly impaired blastocyst development. As no clear candidate emerged from this targeted screen, we focussed on threonine dehydrogenase (TDH), which catalyses threonine catabolism. TDH mRNA and protein was present at similar levels in trophectoderm (TE) and ICM but absent from several adult somatic tissues. We then treated morulae with an inhibitor (Qc1) that blocks TDH from catabolising threonine. Continuous exposure to Qc1 reduced total and high-quality blastocyst development from 37% to 26% and 18% to 8%, respectively (P<0.005). This was accompanied by ∼2-fold decrease in ICM, TE and total cell numbers (P<0.005), which was due to increased autophagy (P<0.05). At the same time, ICM-(NANOG) and TE-restricted (KRT8) genes were up-and down-regulated, respectively (P<0.05). In summary, bovine blastocyst viability depended on TDH-mediated threonine catabolism. However, ICM and TE cells did not metabolically differ in this regard, highlighting species-specific connections between metabolism and pluripotency regulation in mouse vs cattle.

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.

scholarly journals PTPN11 (SHP2) Is Indispensable for Growth Factors and Cytokine Signal Transduction During Bovine Oocyte Maturation and Blastocyst Development

Cells ◽  
2019 ◽  
Vol 8 (10) ◽  
pp. 1272 ◽  
Author(s):  
Muhammad Idrees ◽  
Lianguang Xu ◽  
Seok-Hwan Song ◽  
Myeong-Don Joo ◽  
Kyeong-Lim Lee ◽  
...  
Keyword(s):  
Growth Factors ◽  
Embryo Development ◽  
Oocyte Maturation ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Specific Inhibitor ◽  
Mrna Translation ◽  
Bovine Oocyte ◽  
Blastocyst Development

This study was aimed to investigate the role of SHP2 (Src-homology-2-containing phosphotyrosine phosphatase) in intricate signaling networks invoked by bovine oocyte to achieve maturation and blastocyst development. PTPN11 (Protein Tyrosine Phosphatase, non-receptor type 11) encoding protein SHP2, a positive transducer of RTKs (Receptor Tyrosine Kinases) and cytokine receptors, can play a significant role in bovine oocyte maturation and embryo development, but this phenomenon has not yet been explored. Here, we used different growth factors, cytokines, selective activator, and a specific inhibitor of SHP2 to ascertain its role in bovine oocyte developmental stages in vitro. We found that SHP2 became activated by growth factors and cytokines treatment and was highly involved in the activation of oocyte maturation and embryo development pathways. Activation of SHP2 triggered MAPK (mitogen-activated protein kinases) and PI3K/AKT (Phosphoinositide 3-kinase/Protein kinase B) signaling cascades, which is not only important for GVBD (germinal vesical breakdown) induction but also for maternal mRNA translation. Inhibition of phosphatase activity of SHP2 with PHPS1 (Phenylhydrazonopyrazolone sulfonate 1) reduced oocytes maturation as well as bovine blastocyst ICM (inner cell mass) volume. Supplementation of LIF (Leukemia Inhibitory Factor) to embryos showed an unconventional direct relation between p-SHP2 and p-STAT3 (Signal transducer and activator of transcription 3) for blastocyst ICM development. Other than growth factors and cytokines, cisplatin was used to activate SHP2. Cisplatin activated SHP2 modulate growth factors effect and combine treatment significantly enhanced quality and rate of developed blastocysts.

109 EFFECTS OF LIPOLYTIC AGENTS FORSKOLIN, EPINEPHRINE AND CAFFEINE ON EMBRYONIC DEVELOPMENT AND LIPID CONTENT OF BOVINE EMBRYOS PRODUCED IN VITRO

2009 ◽  
Vol 21 (1) ◽  
pp. 154 ◽  
Author(s):  
M. Barcelo-Fimbres ◽  
G. E. Seidel
Keyword(s):  
Amino Acids ◽  
Fatty Acid ◽  
Embryonic Development ◽  
Lipid Content ◽  
Lipid Accumulation ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Nile Red ◽  
Essential Amino Acids

The objective of this experiment was to evaluate lipid accumulation and embryonic development of bovine morulae treated with different chemicals. A total of 2619 slaughterhouse oocytes from heifers and mature cows were matured in CDM medium (similar to SOF) plus 0.5% fatty acid-free BSA and hormones (M-CDM) for 23 h at 38.5°C in 5% CO2 in air. Frozen–thawed sperm were centrifuged through a Percoll gradient and co-cultured with matured oocytes for 18 h in F-CDM (CDM+heparin). Zygotes were cultured at 38.5°C in 5% CO2/5% O2/90% N2 in CDM-1 with nonessential amino acids, 10 μm EDTA, 0.5% fatty acid free BSA, and 0.5 mm fructose. After 60 h, resulting 8-cell embryos were cultured 120 h in CDM-2 (CDM-1+essential amino acids and 2 mm fructose). A factorial design was used with 7 treatments, 2 ovary sources (cows v. heifers), and 3 bulls (A, B and C) replicated twice for each bull (6 replicates). At Day 2.5 embryo cleavage and 8-cell rates were evaluated, and on Day 6 a total of 755 morulae were randomly assigned to the 7 treatments (control, 2 and 8 mm caffeine, 1 and 4 μm epinephrine, and 10 and 40 μm forskolin). To quantify lipid accumulation, Day 7 blastocysts were fixed and stained with 1 μg mL–1 Nile red dye, after which a digital photograph of the equatorial part of the embryo (including the inner cell mass) was taken at 200×, and fluorescence intensity was measured with Image Pro software from 0 to 255 shades for each pixel (0 = no lipids; 255 = greatest lipid accumulation), as previously reported (Biol. Reprod. 2007 (Suppl. 1), 87–88). Data were analyzed by ANOVA. No differences in cleavage rates (75 v. 68 ± 3.6%) or eight cell rates (61 ± v. 57 ± 2.8%) were found for heifer v. cow oocytes (P > 0.1); however, blastocyst rates per oocyte and per 8-cell embryo were greater for cows than heifers (20 v. 10 ± 2.1%, and 68 v. 35 ± 3.8%, respectively; P < 0.05). Treatments: 2 and 8 mm caffeine produced fewer blastocysts per morula than 1 and 4 μm epinephrine, 10 and 40 μm forskolin and the control (39, 5 v. 54, 49, 48, 54 and 52 ± 5.8%; respectively) (P < 0.01). More lipid content was found in whole embryos and trophoblast of heifer-derived than cow blastocysts (P < 0.05), and forskolin resulted in less lipid content than control, caffeine- and epinephrine-treated morulae in whole embryos, embryonic mass and trophoblasts (P < 0.05; Table 1). In conclusion, mature cows were a better source of oocytes than feedlot heifers for embryonic development. High doses of caffeine were detrimental to embryos, and the addition of the lypolitic agent forskolin reduced lipid content relative to control, caffeine and epinephrine-treated embryos. Table 1.Main effect treatment means of lipid content (arbitrary fluorescence units)

Bovine blastocyst diameter as a morphological tool to predict embryo cell counts, embryo sex, hatching ability and developmental characteristics after transfer to recipients

2006 ◽  
Vol 18 (5) ◽  
pp. 551 ◽  
Author(s):  
Michael Hoelker ◽  
Friedrich Schmoll ◽  
Hendrik Schneider ◽  
Franca Rings ◽  
Markus Gilles ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryo Cell ◽  
Cell Counts ◽  
Bull Calves ◽  
Embryo Sex ◽  
Developmental Characteristics ◽  
Number Of Cells ◽  
Bovine Blastocyst

The aim of the present study was to explore whether the blastocyst diameter and the zona thickness at 168 h after fertilisation are useful parameters to predict quality and viability of bovine in-vitro-produced (IVP)-embryos. Although significant (P < 0.05), the blastocyst diameter at 168 h correlated only poorly with the total number of cells (R2 = 0.13) and with the number of trophectoderm (TE) cells (R2 = 0.17). Hatched blastocysts (n = 66) at 216 h had a significantly greater mean diameter at 168 h (194.8 ± 16.8 µm) compared with either blastocysts that had started but not finished hatching at 216 h (n = 26, 178.4 ± 16.7 µm) or failed to commence hatching (n = 136, 162.7 ± 12.9 µm). Transfer of 101 IVP blastocysts to synchronised recipients resulted in the birth of 38 calves (38%). There were significantly more bull calves born than cow calves (P < 0.05), but this was not correlated with blastocyst diameter or zona thickness at 168 h. There was also no correlation between the diameter of blastocysts or the zona thickness at 168 h and parameters of subsequent developmental characteristics, including rates of pregnancy, resorptions and abortions, pregnancy duration, delivery to term and birthweight. Overall, the present results indicate that the blastocyst diameter and the zona thickness at 168 h are good predictors for subsequent hatching ability in vitro, but not for the number of TE cells, inner cell mass cells or total cells and neither for subsequent developmental characteristics after transfer to recipients.

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&gt;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&lt;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.

scholarly journals Leukemia Inhibitory Factor Stimulates Primitive Endoderm Expansion in the Bovine Inner Cell Mass

2021 ◽  
Vol 2 ◽  
Author(s):  
Lydia K. Wooldridge ◽  
Alan D. Ealy
Keyword(s):  
Leukemia Inhibitory Factor ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Number ◽  
Inhibitory Factor ◽  
Primitive Endoderm ◽  
Total Cell Number ◽  
Cell Numbers ◽  
Bovine Blastocyst

Previous work determined that bovine interleukin-6 (IL6) increases inner cell mass (ICM), primitive endoderm (PE), and total cell number in in vitro produced (IVP) bovine blastocysts. Another IL6 family member, leukemia inhibitory factor (LIF), has the potential to produce the same effects of IL6 due to the presence of its receptor in bovine blastocysts. We compared the abilities of LIF and IL6 to increase ICM cell numbers in day 7, 8, and 9 IVP bovine blastocysts. Supplementation with 100 ng/ml LIF from day 5 onward improved blastocyst formation rates on days 7 and 8 similar to what was observed when supplementing 100 ng/ml IL6. However, LIF supplementation did not cause an increase in ICM numbers like was observed after supplementing IL6. On day 9, increases in PE cell numbers were detected after LIF supplementation, but 300 ng/ml LIF was required to achieve the same effect on PE numbers that was observed by providing 100 ng/ml IL6. Collectively, these results show that LIF can mimic at least some of the effects of IL6 in bovine blastocyst.

The Contribution of Caseins to the Amino Acid Supply for Lactococcus lactis Depends on the Type of Cell Envelope Proteinase

1998 ◽  
Vol 64 (6) ◽  
pp. 1991-1996 ◽  
Author(s):  
Benedicte Flambard ◽  
Sandra Helinck ◽  
Jean Richard ◽  
Vincent Juillard
Keyword(s):  
Amino Acids ◽  
Growth Rate ◽  
Amino Acid ◽  
Lactococcus Lactis ◽  
Cell Envelope ◽  
Essential Amino Acids ◽  
Genetically Engineered ◽  
Amino Acid Requirements ◽  
Rate Limiting

ABSTRACT The ability of caseins to fulfill the amino acid requirements ofLactococcus lactis for growth was studied as a function of the type of cell envelope proteinase (PI versus PIII type). Two genetically engineered strains of L. lactis that differed only in the type of proteinase were grown in chemically defined media containing αs1-, β-, and κ-caseins (alone or in combination) as the sources of amino acids. Casein utilization resulted in limitation of the growth rate, and the extent of this limitation depended on the type of casein and proteinase. Adding different mixtures of essential amino acids to the growth medium made it possible to identify the nature of the limitation. This procedure also made it possible to identify the amino acid deficiency which was growth rate limiting for L. lactis in milk (S. Helinck, J. Richard, and V. Juillard, Appl. Environ. Microbiol. 63:2124–2130, 1997) as a function of the type of proteinase. Our results were compared with results from previous in vitro experiments in which casein degradation by purified proteinases was examined. The results were in agreement only in the case of the PI-type proteinase. Therefore, our results bring into question the validity of the in vitro approach to identification of casein-derived peptides released by a PIII-type proteinase.

58 ISOLATION OF BOVINE TROPHOBLAST AND ITS REPROGRAMMING BY NUCLEAR TRANSFER

2011 ◽  
Vol 23 (1) ◽  
pp. 134
Author(s):  
I. M. Saadeldin ◽  
B. H. Kim ◽  
B. Roibas da Torre ◽  
O. J. Koo ◽  
G. Jang ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Inner Cell Mass ◽  
Quantitative Polymerase Chain Reaction ◽  
Cell Mass ◽  
Donor Cell ◽  
Developmental Competence ◽  
Control Group ◽  
Embryonic Cells ◽  
Blastocyst Development

Nuclear transfer (NT) has been used to produce many cloned offspring using several types of cells, including embryonic cells. Even though inner cell mass cells have been used as donor karyoplast for producing cloned animals, there are few studies using trophoblast. In mice, clones were born by nuclear transfer of trophoblasts from the expanded blastocyst into enucleated oocytes as a trial to show the totipotency of both inner cell mass and trophectoderm cells isolated from blastocysts (Tsunoda and Kato 1998 J. Reprod. Fertil. 113, 181–184). However, bovine trophoblast cell (TC) lines have not been used in NT to date. The purpose of this study was to elucidate whether TC as donor cell can be reprogrammed in bovine enucleated oocyte and determine the relative abundance of interferon tau (IFNτ) expression in the resulting cloned preimplantational embryos. Hatched blastocysts produced by IVF were used to isolate TCs on mouse embryonic fibroblasts treated with mitomycin C as feeder cells. TCs and adult fibroblasts (AF, control group for NT) were microinjected to perivitelline space of in vitro mature enucleated oocytes and electrically fused. Reconstructed embryos were chemically activated and cultured in a 2-step chemically defined medium. Levels of IFNτ expression in IVF-, TC-, and AF-derived blastocysts were analyzed using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). IVF produced embryos were used as reference to analyze the linear progressive expression of IFNτ through mid-, expanded, and hatching blastocysts. As a result, TCs expressing IFNτ were successfully isolated and cultured on feeder layers. It grew as cell sheets of cuboidal epithelium with high proliferation capacity as a single colony originated from a small clump of cells measured 0.5 cm within 7 days of culture. TCs were reprogrammed in the enucleated oocytes to blastocyst with similar efficiency to AF (14.5% and 15.6%, respectively; P ≤ 0.05). RT-qPCR studies showed that IFNτ expression was higher in TC-derived blastocysts than IVF- and AF-derived blastocysts. Both IVF- and TC-derived blastocysts, showed progressive increase of IFNτ expression through the advancement of blastocyst development when it was compared to AF-derived blastocysts. In conclusion, using TCs expressing IFNτ as donor cell for bovine NT could increase the developmental competence of cloned embryos as indicated by progressive linear increase in IFNτ expression. This study was supported by grants from IPET (#109023-05-1-CG000), NRF (#M10625030005-10N250300510), MKE (#2009-67-10033839, #2009-67-10033805), and BK21 program. Saadeldin I. M. is supported by Islamic Development Bank (IDB) merit scholarship, Jeddah, Saudi Arabia.

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.

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