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

Abstract

Equine embryos develop in vitro in 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 O16 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 . 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 for POU5F1. 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 in in vitro-fertilized equine blastocysts. These findings expand our understanding of the factors that affect embryo development in the horse.

Altered morphokinetics in equine embryos from oocytes exposed to DEHP during IVM

“…The semen was extended with INRA 96 (IMV Technologies, Piacenza, Italy) at the concentration of 20–25 × 10 6 sperm cells/ml, and delivered to the laboratory on the same day for use. Sperm were prepared by the swim‐up procedure as reported by Choi et al () and Choi, Chung, Walker, Westhusin, and Hinrichs (). In detail, semen was layered (0.2 ml/tube) in two conical Falcon tubes under 1 ml of pre‐heated Sperm‐Chatot, Ziomet and Bavister medium (Sp‐CZB) and incubated in tilted position at 38.2°C for 20 min under 5% CO 2 in air.…”

“…The Piezo‐drill ICSI procedure was performed as previously reported by Choi et al () with some modifications. For its use in ICSI, 0.5 μl of the sperm suspension obtained as described above was placed on the surface of a 5‐μL droplet of Sp‐CZB containing 7% (w/v) polyvinylpyrrolidone (PVP‐360) and motile sperm cells were identified after swimming down into the droplet.…”

“…On the same day, embryos morphologically classified as blastocysts underwent a triple‐staining procedure for concurrent evaluation of nuclear chromatin, mitochondria (mt) and ROS as described below. The blastocyst stage was confirmed if they contained more than 64 cells and had begun the organization of outer presumptive trophoblast cells (Choi et al, ).…”

Effect of intra-ovarian injection of mesenchymal stem cells in aged mares

“…In aged mares after MSC injection, the aspirate from each ovary was filtered separately, and the granulosa cells from the aspirates were flash-frozen for genotyping of nuclear microsatellite markers and mitochondrial DNA sequencing as described below. In vitro maturation and intracytoplasmic sperm injection of recovered oocytes Recovered oocytes were matured in vitro as previously described [27]. Briefly, oocytes were held overnight [28] then transferred to maturation medium (M199 with Earle's salts, 5 mU/ml FSH (Sioux Biochemicals, Sioux Center, IA), 10% FBS, and 25 μg/ml gentamicin) and cultured for 24-30 h at 38.2°C in a humidified atmosphere of 5% CO 2 in air.…”

“…Intracytoplasmic sperm injection was performed as previously described [27] using a piezo drill. Presumptive zygotes were cultured in a commercial human embryo culture medium (GB; Global medium, LifeGlobal, Guilford, CT) supplemented with 10% FBS at 6% CO 2 , 5% O 2 , and 89% N 2 at 38.2°C.…”