Effect of clinically-related factors on in vitro blastocyst development after equine ICSI

Equine intracytoplasmic sperm injection (ICSI) is being used clinically for foal production, but little information is available on factors affecting the efficiency of this procedure. We examined factors that may influence blastocyst development when ICSI is performed clinically, i.e., on oocytes recovered from live mares by transvaginal ultrasound-guided follicle aspiration (TVA), using sperm from the stallion of the client's choice. In a clinical setting, there may be a delay from the time of TVA to isolation of oocytes from the aspirated fluid. In a preliminary study, oocytes from fluid held for 1.5 h at ambient temperature (26°C-33°C) yielded 32% blastocysts; however, in experiment 1, fluid held at 32 °C for 2 h after aspiration yielded 16% blastocysts versus 23% for aspirates processed immediately. Performing TVA/ICSI throughout the year would increase production from valuable mares, but efficiency during the nonbreeding season is unknown. In addition, to reduce the possibility of infection after TVA, administration of antibiotics to the mare before TVA is indicated; however, these could affect oocyte quality. In experiment 2, follicle numbers at the time of TVA were significantly higher in December to January than for the same mares during the breeding season. Oocyte recovery rates on TVA were 60% to 66% and the blastocyst rate was 18%. An equivalent blastocyst rate (18%) was achieved after administration of ampicillin and gentamicin to mares before TVA. In experiment 3, we verified that stallion differences exist in rates of cleavage after ICSI with motile sperm. In sperm from a low-performing stallion, density-gradient centrifugation followed by swim-up was associated with significantly higher rates of cleavage (45% vs. 18%) and blastocyst development (14% vs. 0%) than those for density gradient alone. In experiment 4, parthenogenetic activation with ionomycin and 6-dimethylaminopurine yielded 40% blastocysts. Frozen-thawed sperm that were immotile after nitrogen tank failure did not produce blastocysts; exogenous activation after ICSI increased cleavage rate but did not yield blastocysts. These studies provide information on factors that may affect in vitro blastocyst formation after equine ICSI as it is performed in a clinical program.