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Effects of different oocyte retrieval and in vitro maturation systems on bovine embryo development and quality.

Zygote (Cambridge, England) (2014-01-16)
Sandra Milena Bernal Ulloa, Julia Heinzmann, Doris Herrmann, Bernd Timmermann, Ulrich Baulain, Rudolf Großfeld, Mike Diederich, Andrea Lucas-Hahn, Heiner Niemann
ABSTRACT

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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Forskolin, from Coleus forskohlii, ≥98% (HPLC), powder
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Glycerol, BioReagent, suitable for cell culture, suitable for insect cell culture, suitable for electrophoresis, ≥99% (GC)
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Glycerol, ≥99.5%
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Glycerol, BioXtra, ≥99% (GC)
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Glycerol, for molecular biology, ≥99.0%
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Cilostamide, phosphodiesterase inhibitor
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Forskolin, For use in molecular biology applications
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Triton X-100, laboratory grade
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Propidium iodide solution, solution (1.0 mg/ml in water)
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Sodium bicarbonate-12C, 99.9 atom % 12C
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Glycerol, FCC, FG
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Glycerol, BioUltra, for molecular biology, anhydrous, ≥99.5% (GC)
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Glycerol, Vetec, reagent grade, 99%
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3-Isobutyl-1-methylxanthine, ≥99% (HPLC), powder
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3-Isobutyl-1-methylxanthine, ≥99%, BioUltra
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Sodium bicarbonate, Hybri-Max, powder, suitable for hybridoma, ≥99.5%
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Sodium bicarbonate, powder, BioReagent, for molecular biology, suitable for cell culture, suitable for insect cell culture
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Sodium bicarbonate, BioXtra, 99.5-100.5%
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Glycerin, meets USP testing specifications
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Propidium iodide, ≥94.0% (HPLC)
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Hypotaurine, ≥98% (TLC)
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Formaldehyde solution, for molecular biology, 36.5-38% in H2O
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Glycerol solution, 83.5-89.5% (T)
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Formaldehyde solution, ACS reagent, 37 wt. % in H2O, contains 10-15% Methanol as stabilizer (to prevent polymerization)
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Formaldehyde-12C solution, 20% in H2O, 99.9 atom % 12C
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Formaldehyde solution, meets analytical specification of USP, ≥34.5 wt. %
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Formaldehyde solution, for molecular biology, BioReagent, ≥36.0% in H2O (T)
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Sodium bicarbonate, Vetec, reagent grade, 99%
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Sodium bicarbonate, suitable for cell culture