177 Equine Sperm Decondensation and Blastocyst Formation After Conventional versus Piezo-Driven Intracytoplasmic Sperm Injection

Abstract

Intracytoplasmic sperm injection (ICSI) is currently the most effective method for in vitro fertilization in the horse. There are 2 main techniques, conventional (Conv) ICSI and piezo-driven (Piezo) ICSI. Many laboratories reporting good equine blastocyst rates (>20% per injected oocyte) use Piezo ICSI, but it is not known whether the Piezo confers an advantage. We compared sperm decondensation and blastocyst formation between the 2 techniques. A blunt, 6-µm inner diameter needle, loaded with 10 µL of Fluorinert was used for Piezo ICSI; 1 pulse was used to penetrate the oolemma. Frozen-thawed semen from one stallion was used. In experiment 1, in vitro-matured equine oocytes were randomly assigned to Conv or Piezo ICSI, performed concurrently by separate operators. Blastocyst formation was evaluated on Days 7 to 10 and confirmed by DAPI staining. Data were analysed by Fisher’s exact test. There was no significant difference in blastocyst rates (32/82, 39% for Conv and 35/87, 40% for Piezo; P > 0.1). In experiment 2, equine sperm head decondensation after ICSI was evaluated using porcine oocytes, due to scarcity of equine oocytes. Porcine oocytes were recovered from slaughterhouse-derived ovaries and matured in vitro using a biphasic maturation culture system. Mature oocytes were subjected to Conv or Piezo ICSI, using equine sperm treated with a mitochondrial stain to allow identification of the sperm tail. The oocytes were fixed in 4% paraformaldehyde immediately after injection (0 h) or were cultured for 3, 6, 9, or 18 h after injection, and then fixed. Fixed oocytes (15-22 per treatment per period) were stained with DAPI and the area of the sperm head, in arbitrary units, determined using ImageJ software (National Institutes of Health, Bethesda, MD, USA). The medians were compared using the Mann-Whitney U-test. Sperm heads in the Piezo treatment increased in area over time, from a median of 1242 at 0 h to 54,991 at 18 h. In contrast, sperm heads in the Conv treatment largely failed to decondense, having median areas of 1275 at 0 h and 1510 at 18 h. Sperm head area was significantly greater for sperm in the Piezo than in the Conv treatment for all time periods except 0 h (P < 0.05). Because this conflicted with the blastocyst results obtained with horse oocytes, we conducted experiment 3 to examine sperm head decondensation after ICSI in horse oocytes. Oocytes (8 to 12 per treatment per period) were fixed 0, 6, or 18 h after ICSI. There was no difference between techniques in sperm head area at any time (median values for 0, 6 and 18 h of 1280, 4323, and 57,185 respectively for Piezo and 1326, 1604, and 62,558 for Conv; P > 0.2). These results indicate that there is a species-specific difference in processing of sperm after ICSI, dependent on injection technique. Further work evaluating sperm from additional stallions, as well as porcine sperm, is necessary to determine whether sperm source affects these results.

Research supported by the Clinical Equine ICSI Program, Texas A&M University.