H. S. Canesin

Effect of age and endometrial degenerative changes on uterine blood flow during early gestation in mares

The present experiment was divided into three studies to investigate the effect of age and endometrial degeneration on uterine blood flow of mares. In study 1, the influence of semen infusion and conception was evaluated using noninseminated (non-AI), inseminated nonpregnant (AI-NP), and inseminated pregnant (AI-P) mares (n = 7 mares/group). In study 2, the effect of age was investigated using young (≤6 years), adult (from 8 to 12 years), and old (≥15 years) pregnant mares (n = 7 mares/group). In study 3, uterine blood flow was also characterized in pregnant mares (n = 7 mares/group) with minimal, moderate, or severe endometrial degenerative changes (GI, GII, and GIII, respectively). Uterine vascular perfusion and pulsatility index from mesometrium attachment arteries were recorded daily from Day 0 (day of ovulation) to Day 12 and between Days 0 and 20 in nonpregnant and pregnant mares. Analysis according to the position of the embryo (uterine horn with embryo vs. opposite horn) was performed from Day 12 until Day 20. In study 1, increased uterine vascular perfusion and decreased pulsatility index were detected (P < 0.001) between Days 3 and 5 in non-AI and AI-P mares, whereas the uterine vascular perfusion of AI-NP mares did not change (P > 0.05) throughout the experiment. In study 2, the vascular perfusion of the uterine horn with embryo was higher (P < 0.001) than in the opposite uterine horn from Day 12 until Day 20 in both young and adult mares. With exception of Day 15, both uterine horns of old mares showed similar (P > 0.1) vascular perfusion from Day 12. In study 3, vascular perfusion of both uterine horns was lower (P < 0.001) in GIII mares than in GI and GII mares. After Day 15, the scores for uterine vascular perfusion were consistently greater (P < 0.001) in the uterine horn with embryo than in the opposite horn in GI mares. Results showed, for the first time, the reduced blood flow of the gravid uterus during early gestation in older mares and in mares with significant endometrial degeneration.

Vitrification of germinal-vesicle stage equine oocytes: Effect of cryoprotectant exposure time on in-vitro embryo production

Previous studies have found low rates of blastocyst development (0-11%) after vitrification of germinal vesicle (GV)-stage equine oocytes. In this study, we systematically evaluated a short (non-equilibrating) system for GV-stage oocyte vitrification. In Exp. 1, we assessed oocyte volume in cumulus-oocyte complexes (COCs) exposed to components of a short protocol, using 2% each of ethylene glycol and propylene glycol in the first solution (VS1); 17.5% of each plus 0.3 M trehalose in the second solution (VS2); and fetal bovine serum as the base medium. Based on the time to oocyte minimum volume, we selected a 40-sec exposure to VS1. In Exp. 2, we evaluated exposure times to VS2 and, based on rates of subsequent maturation in vitro, we selected 65 s. In Exp. 3, we used the optimized vitrification system (40-VS1; 65-VS2) and evaluated three warming procedures. Blastocyst development after ICSI was equivalent (15%) for COCs warmed in either standard (trehalose stepwise dilution) or isotonic (base medium) solutions, but was reduced (0%) for COCs warmed in a highly hypertonic (1.5 M trehalose) solution. Exposure to the vitrification and warming solutions, without actual vitrification, was associated with reduced blastocyst development (0-5%; Exp. 4). We conclude that this optimized short protocol supports moderate blastocyst production after vitrification of GV-stage equine COCs. Oocytes can be warmed in isotonic medium, which simplifies the procedure. The systems used still showed a high level of toxicity and further work is needed on both vitrification and warming methods to increase the efficiency of this technique.