Effect of Disaccharide Inclusion in Vitrification and Warming Solutions on Developmental Competence of Vitrified/Warmed Germinal Vesicle Stage Buffalo Oocytes.

BACKGROUND: Cryopreservation of immature oocyte is a potential strategy for preserving the female germline, providing a non-seasonal, easily accessible source for reproduction and science. Exposure of oocytes to high concentrations of cryoprotectants during vitrification is toxic and can negatively impact the fertilization ability and development of vitrified/warmed oocytes. OBJECTIVE: 1) to evaluate the effects of exposure of buffalo germinal vesicle (GV) oocytes to different vitrification solutions (VS), either supplemented with or without sucrose, on cumulus expansion and nuclear maturation following IVM; and 2) to compare the effects of sucrose and trehalose in the warming solution on developmental competence of buffalo oocytes vitrified at the GV-stage. MATERIALS AND METHODS: Cumulus oocyte complexes (COCs) obtained at slaughter from mature buffalo ovaries were randomly assigned into five groups: control - directly subjected to IVM); VS1 group - exposed to 20% ethylene glycol (EG) + 20% glycerol (GLY) + 0.5 M sucrose; VS2 group - exposed to 20% EG + 20% GLY; VS3 group - subjected to 20% EG+20% dimethyl sulfoxide (DMSO) + 0.5 M sucrose; and VS4 group - subjected to 20% EG+20% DMSO. Following cryoprotectant dilution, viable oocytes were matured in vitro for 22 h; cumulus expansion and nuclear maturation were then evaluated (Experiment 1). COCs were vitrified by solid surface vitrification (SSV) in a solution composed of 20% EG + 20% DMSO (VS4). Following vitrification, COCs were warmed in a solution composed of either sucrose or trehalose in decreasing concentrations (1 M, 0.5 M and 0.25 M). Morphologically viable oocytes were matured, fertilized and cultured in vitro. Cleavage and blastocyst rates were evaluated at 30 h and day 7 post-insemination (p.i.), respectively (Experiment 2). RESULTS: Exposure of GV-buffalo oocytes to different cryoprotectant combinations did not significantly affect cumulus expansion following IVM. However, nuclear maturation rate (oocytes at MII) was significantly higher (P<0.05) in the groups exposed to sucrose-free vitrification solutions (VS2 and VS4) and not significantly different from the control. Compared with the control group, the cleavage and blastocyst rates were significantly (P<0.05) lower in oocytes vitrified and then warmed in a solution containing trehalose; whilst this was not the case when sucrose was present in the solution. CONCLUSION: Our results suggest that exposure of buffalo GV-oocytes to sucrose-free vitrification solutions improved nuclear maturation after IVM. Moreover, warming of vitrified buffalo oocytes in sucrose-based solution improved preimplantation development following IVM and IVF compared to trehalose based media.

Plasma free tryptophan revisited: what you need to know and do before measuring it.

Plasma free tryptophan (Trp) is an important peripheral parameter widely used by psychopharmacologists to assess Trp entry into the brain for cerebral serotonin synthesis, although, along with total Trp, it can give much more information on Trp metabolism and disposition. Plasma free Trp is, however, a labile parameter easily influenced by a great many modulators, including fasting, food intake, many prescribed and over the counter medications, consumption of alcoholic and of common hot beverages, illicit drug use, some hormones, exercise and mild stressors. Interpretation of changes in plasma free Trp requires appropriate preparation of ultrafiltrates from freshly isolated plasma or serum, accurate analytical methodology and awareness of the multitude of physiological and pharmacological modulators of its concentration. This article highlights these points and makes recommendations aimed at avoiding pitfalls in studies involving this parameter.

Application of the Phenomenex EZ:faasttrade mark amino acid analysis kit for rapid gas-chromatographic determination of concentrations of plasma tryptophan and its brain uptake competitors.

The Phenomenex EZ:faast amino acid analysis kit is available for gas (GC) or liquid (LC) chromatographic analysis of amino acids (AA) using mass spectrometry (MS) and other GC detectors. We used it for rapid GC determination of plasma tryptophan, its brain uptake competitors (Val, Leu, Ile, Phe and Tyr) and many other amino acids. Based on solid-phase extraction, this fast method enables one person to process two plasma samples in 8-10 min and six samples in approximately 15 min up to GC injection and a 7-min GC run per plasma sample. Using a Perkin-Elmer Clarus 500 GC, a Total Chrome software, a flame-ionisation detector (FID) and norvaline as internal standard, we used this method to analyse approximately 1,000 plasma samples from normal subjects undergoing acute tryptophan depletion and loading tests. The limit of detection for most amino acids is 1 nmol/ml (1 microM) and in many cases less. With manual injection, coefficients of variation for the above six amino acids were 1.5-6.2% (intra-assay) and 3.8-9.7% (inter-assay). This simple, rapid and elegant method will be valuable to the amino acid analyst and researcher, as it can save much manpower time and meet urgent emergency requests and the demands of a high-throughput laboratory.